Angiogenic and immunoglobic applications of anti cd160 specific compounds obtainable from mab cl 1-r2

ABSTRACT

The present invention relates to biological and medical application of an anti-CD160 monoclonal antibody (CL1-R2 CNCM I-3204) an of the conservative equivalents thereof. It more particularly relates to the applications of these anti-CD 160 compounds in the fields of EC angiogenesis, and NK and T cytokine production

FIELD OF THE INVENTION

The present invention relates to an anti-CD160 specific mAb (CL1-R2accessible under hybridoma deposit number CNCM I-3204), to anti-CD160specific compounds deriving therefrom, and to the biological and medicalapplications of such anti-CD160 compounds and mAb.

BACKGROUND OF THE INVENTION

The present invention more particularly relates to means forspecifically controlling and regulating:

-   -   the angiogenesis of endothelial cells (EC), and    -   the contribution of NK and T cells to the regulation of the        immune system.

The different aspects of the invention share the common feature ofimplementing or requiring an anti-CD160 monoclonal antibody (mAb) of thepresent invention, namely the mAb referred to by the inventors as CL1-R2(hybridoma Budapest Treaty deposit CNCM I-3204), or a conservativeequivalent thereof.

The present invention indeed demonstrates that the receptor CD160(previously also referred to as BY55), which is known to be expressed bycytotoxic NK and T subsets (CD56^(dim) CD16^(bright) CD3⁻ NK; T CD8+;TCRγδ), is involved in both angiogenesis and immune system regulation.CD160 structure has been extensively described in prior art documents,see e.g. WO 98/21240 in the name of the DANA-FARBER CANCER INSTITUTE.

EC and Angiogenesis:

Angiogenesis, the formation of new capillaries from the preexistingblood vessels, is a crucial component of embryonic vascular developmentand differentiation, wound healing, and organ regeneration. It howeveralso contributes to the progression of pathologies that depends onneovascularization, including tumor growth, diabetes, ischemic oculardiseases, and rheumatoid arthritis (Risau, 1997; Ferrara, 1997). Whilethe most important mediators of angiogenesis, the vascular endothelialcell growth factor (VEGF) family and fibroblast growth factor family arewell define, angiogenesis stands as a complex process involving multiplegene products expressed by different cell types all contributing to anintegrated sequence of events.

WO 03/018048 in the name of ABTECH et al. relates to the use of twosoluble HLA Class I molecules, namely sHLA-G1 and sHLA-B7, to inhibitangiogenesis or to detect angiogenic sites. Supportive to thisanti-angiogenic effect is the demonstration that sHLA-G1 inhibitsendothelial cells (EC) proliferation and migration. It is also shownthat sHLA-G1 and sHLA-B7 may inhibit the progression of a tumor inducedby grafting human prostate adenocarcinoma cells in nude mice.

WO 03/018048 also mentions that an anti-CD160 antibody referred to asCL1-R2 inhibits the action exerted by sHLA-G on EC migration. It istherefrom deduced that BY55 could be an endothelial receptor for sHLA-G(cf. WO 03/018048 as published, page 23 lines 3-8).

The skilled person would however notice that WO 03/018048 gives nopublicly available source for the mentioned CL1-R2 antibody.

On the other hand, soluble HLA, such as sHLA-G1 and sHLA-B7, are naturalligands for numerous receptors.

Hence, there remains a need in prior art for means that would besufficiently specific to the angiogenesis signaling pathways to enablethe elucidation of the mechanisms they involve. Specificity is alsoneeded to provide medically useful compounds that can exert a specificcontrol on angiogenesis without necessarily disturbing other signalingpathways.

NK and T Cells and the Immune System:

NK cells constitute a subset of lymphocytes that play a role in innateimmunity directed against virally-infected or tumor cells. Theireffector functions are the killing of target cells and cytokineproduction. NK cells use a combination of inhibitory and activatingreceptors expressed at their cell surface to mediate target cell killingand cytokine release upon interaction with specific ligands. Uponspecific engagement with these ligands present on target cells, theyrelease cytolytic granules containing perform and granzyme thatcontribute to target cell apoptosis. Upon contact with sensitive targetcells, they also produce a number of cytokines, including IFN-γ, TNF-αand GM-CSF early in the innate immune response that modulate adaptiveimmunity by regulating T cell function. The release of IFN-γ by NK cellsin both inflamed tissues and secondary lymphoid organs influence thedendritic cells-initiated adaptive immune response. IFN-γ secreted byuterine NK cells may also control placental development andvascularisation during pregnancy.

Yet, only few human activating NK cell receptors have been shown toinduce cytokine production upon specific engagement.

KIR2DL4 (CD158d) induces IFN-γ, production in resting and activated NKcells. CD16 is a low-affinity FcγRIII receptor responsible forAb-dependent cellular cytotoxicity (ADCC). Signaling via CD16 triggersthe production of cytokines, including IFN-γ, GM-CSF, and severalchemokines. Incubation of activated NK cells with anti-NKp30 oranti-NKp46 mAb led to IFN-γ production by NK cells. Human NKG2Dactivating receptor that recognizes the stress-induced MICA and MICBmolecules as well as the ULBP family of molecules and plays a major rolein NK cell-mediated cytotoxicity is apparently unable to producecytokines once triggered by specific mAbs.

T cells including CD8+ and CD4+ T cells also produce cytokines. Th1cells produce IL-2 and IFN-γ, whereas Th2 cells produce IL-4. Theeffector functions of CD8+ T cells partially overlap those of CD4+ Tcells. Naive T cells can differentiate into at least two subsets withdistinct cytokine patterns: T-cytotoxic 1 cells secrete a Th1-likecytokine pattern, while T cytotoxic 2 cells secrete Th2 cytokines.Currently, it is customary to consider IFN-γ to represent a typical type1 cytokine, whereas the signature cytokine of type 2 response is IL-4.

Cytokines intervene in the differentiation and stimulation ofantibody-producing B cell clones and the cytopathic action of cytotoxicT cells. Likewise, cytokine secretion influences the cell-destroyingcapacity of NK cells, and the capacity of macrophages to phagocytosedifferent bacterial plaque components.

The present invention provides with means for specifically controllingup- or down-regulation of cytokine production. The means specificallyacts on the CD160 signaling pathways.

Among the different activating NK cell receptors described to date,CD160 is the only non-clonally expressed receptor on the majority ofcirculating NK cells. CD160+ cells correspond to the non-proliferating,highly cytolytic, CD56^(dim) CD16⁺ NK subset. CD160 engagement by HLA-Cmolecules mediates cytotoxic function.

CD160 is expressed by circulating CD56^(dim) CD₁₆ ^(bright) CD3⁻ NK,which constitute the majority of PB-NK cells.

CD56^(dim) NK cell subset is more naturally cytotoxic and produces lessabundant cytokines than CD₅₆ ^(bright) subset following activation bymonocytes. CD56^(dim) NK cell subset also expresses a specific patternof chemokine receptors and adhesion molecules. Such phenotype ischaracteristic of terminally differentiated effector cells. CD160⁺ NKcells have a high cytotoxic activity potential, do not proliferate toIL-2, and mediate cell lysis upon interaction with HLA-C.

In contrast to other human NK cell receptors described to date, CD160receptor appears unique for the following reasons. It is encoded by agene located on human chromosome 1, it is a glycosyl phosphatydilinositol (GPI)-anchored molecule and its cell surface expression isdown-modulated by NK cell activation mediated by cytokines includingIL-2 and IL-15. As described for the killer cell Ig-like inhibitoryreceptors, CD160 is also expressed by γδ T cells, and a subset of αβCD8+ T cell.

SUMMARY OF THE INVENTION

The present invention relates to an anti-CD160 monoclonal antibody (mAbCL1-R2 obtainable from hybridoma TM60 accessible under CNCM depositnumber I-3204) and to the conservative anti-CD160 equivalents thereof.

It more particularly relates to the applications of these anti-CD160compounds in the fields of EC angiogenesis, and NK and T cytokineproduction.

The present invention indeed demonstrates that CD160 is expressed byendothelial cells (EC), and the anti-CD160 compounds of the inventioncan act as CD160 activating ligands. Stimulation of the CD160 signalingpathway by the anti-CD160 compounds of the invention induces ananti-angiogenic effect.

The present invention also demonstrate that CD160 is expressed not onlyby the cytotoxic NK and T subsets, but also by CD4+ T cells culturedwith IL-15 (expressing cytotoxic activity), and that CD160 stimulationby aggregated anti-CD160 compounds of the invention leads to cytokineproduction. The cytokine profile that is thus obtained is uniquecompared to those obtained by stimulation of other NK-expressedreceptors. It is also unique in the sense that it is very closelymimicking the cytokine profile induced by CD160 stimulation with naturalligands (membrane bound HLA). These cytokines notably comprise IFN-γ,TNF-α and IL-6. It is the first time that there is provided compoundswhich are not natural ligands, but which can induce IL-6 production fromNK cells. Cytokine production induced by cell membrane HLA molecules canbe inhibited using either the anti-CD160 compounds of the invention insoluble form, or anti-CD160 compounds of the invention which comprise atleast one CD158b binding site in addition to their CD160 bindingsite(s).

The present invention hence encompasses the hybridoma TM60 as such, theCL1-R2 monoclonal antibody (mAb), the anti-CD160 compounds of theinvention, any composition or kit comprising them, and any drugcontaining at least one of them.

The present invention also relates to means enabling the identificationof CD160 ligands, CD160 membrane-associated molecules, and CD160 cytosolsecond messengers.

DETAILED DESCRIPTION

The present invention gives a publicly available source of an anti-CD160specific monoclonal antibody (mAb), which is referred to by theinventors as CL1-R2. A CL1-R2 producing hybridoma has been deposited atthe Collection Nationale de Cultures de Microorganismes C.N.C.M.Institut Pasteur in accordance with the terms of the Budapest Treaty onApr. 28, 2004 (C.N.C.M. Institut Pasteur 25, rue du Docteur Roux F-75724Paris Cedex 15 France). The deposited hybridoma has CNCM deposit numberI-3204. The present invention hence relates to the hybridoma TM60accessible under CNCM deposit number 1-3204, as well as to theanti-CD160 mAb obtainable therefrom (CL1-R2).

The present invention also provides with anti-CD160 compounds obtainablefrom said CL1-R2 mAb, e.g. as CL1-R2 fragments or derivatives.

The inventors further provide demonstrations relating to:

-   -   endothelial cells (EC) and angiogenesis, and to    -   NK and T cells and cytokine production.

These demonstrations share the common feature of implementing saidCL1-R2 mAb or conservative anti-CD160 compounds obtainable therefrom.

EC and Angiogenesis:

The present invention provides the demonstration that CD160, a receptorwhich up to now was known to be expressed by a cytotoxic subset of NKcells and by CD8+ and TCRγδ T cells, is also expressed by endothelialcells (EC) as a membrane receptor, and that CD160 mediates HLAanti-angiogenic signaling.

The present invention also demonstrates that the anti-CD160 mAb that wassaid in WO 03/018048 to inhibit HLA-G action on EC does in fact notinhibit it, but mimics it.

The present invention further demonstrates that the binding of, andpreferably the cross-linking of CD160 by appropriate anti-CD160compounds inhibit the vessel formation and growth that is induced bypro-angiogenic factors such as VEGF or FGF2 on EC. The present inventionthus provides the first direct demonstration that the formation of newcapillaries can actually be regulated and controlled, and also providesindustrially effective means therefor. The present invention henceprovides actual pharmaceutical and medical applications.

Such applications notably include the prevention, symptom alleviation ortreatment of those pathologies or conditions which are due to, orfavored by an activity of neo-vascularization. Under thesecircumstances, neo-vascularization is acting as a pro-pathologiccomponent. The activity of neo-vascularization is then considered torepresent an undesired activity, or to be at an excessive level.

Such neo-vascularization-feeded pathologies or conditions notablycomprise tumor growth (e.g. the growth of tumors), diabetes, ischemicocular diseases, and rheumatoid arthritis. They also includepre-eclampsia or eclampsia, which are characterized by an insufficientblood supply at the fetus-placenta interface (insufficient orinappropriate endovascular trophoblast invasion of maternal spiralarteries).

According to an advantageous aspect of the invention, appropriate meansinclude those anti-CD160 compounds that have an affinity for binding toCD160 that is sufficiently high to compete with CL1-R2 for binding toCD160.

As mentioned above, CL1-R2 is the anti-CD160 mAb that is produced by thehybridoma accessible under CNCM deposit number I-3204.

When it relates to EC-expressed CD160, CL1-R2 can be used in solubleform, as well as in aggregated form. Both forms induce signaltransduction upon binding to CD160 (i.e. transduction of a signal ofangiogenesis inhibition). The aggregated form simply has a higheraffinity for binding to CD160 than the soluble form.

Anti-CD160 mAb have the special technical advantage of having aspecificity that HLA ligands do not have. If they were administered to aliving organism, such as e.g. a human being, HLA ligands would bind toCD160 as well as to many other receptors, and would thereby inducecompletely uncontrolled chain reactions in said organism. On atherapeutic point of a view, HLA ligands hence have no proven industrialapplicability.

Anti-CD160 mAbs such as CL1-R2 are specific of CD160, i.e. they have aCD160 affinity that is sufficient for them to bind essentially only toCD160, at least under in vivo-like conditions. Hence, contrary to HLAligands, anti-CD160 mAbs have industrial applicability as therapeuticagents.

The present invention hence provides for the first time agents which areable to act on CD160 as activating ligands, and which are also usable asagents to be administered in a living organism in need of a CD160activating treatment. In short, the present invention provides the firsttherapeutically-compliant CD160 activating agents.

Such appropriate anti-CD160 candidate ligands of course include CL1-R2itself as well as conservative fragments and derivatives thereof.

An aspect of the present invention also resides in the fact that it nowprovides the demonstration that CD160 is not expressed by tumor cells(Lewis lung carcinoma cells), but that those EC that surrounds orinfiltrates tumors actually express CD160.

The anti-CD160 mAbs as well as conservative fragments and derivatives ofthe invention can be used as therapeutic agents against malignant cellssuch as B-cell chronic lymphocytic leukemia (CLL) which expressed CD160molecules at their cell membrane.

The anti-CD160 compounds of the invention hence are very useful meansfor preventing, treating, or alleviating the symptoms of a tumordevelopment.

The present invention also provides means to identify pharmaceuticallyuseful compounds by screening for binding to CD160, and/or by screeningfor CD160-specific membrane-bound or cytosolic effectors. Such effectorsrepresent useful target for anti-angiogenic therapy.

CD160 belongs to the immunoglobulin supergene family. Descriptiveinformation on CD160 can be found onhttp://www.ncbi.nlm.nih.gov/prow/guide/1660590458_g.htm. The cDNAsequence of human CD160 is described as SEQ ID NO:1 (1361 bp) in WO98/21240 (DANA-FARBER CANCER INSTITUTE).

The mRNA sequence of human CD160 is available from Genbank underAF060981 accession number, the mRNA sequence of mouse CD160 has AF060982Genbank accession number.

The protein sequence of human CD160 is described as SEQ ID NO:2 in saidWO 98/21240, and is also available from Genbank under AAC72302 accessionnumber (181 aa).

CD160 nucleic acids can be isolated from CD160-expressing cellsfollowing any routine procedure that is available to the skilled person[see e.g. the procedures disclosed in Molecular Cloning, A LaboratoryManual (2^(nd) Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor);Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston,More, Feldman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience,NY, N.Y. 1992].

Naturally-occurring CD160-expressing cells can notably be found withincytolytic NK and T cells (such as CD56^(dim) CD16⁺ NK cells and TCRγδand TCRαβ⁺ CD8^(bright) CD95⁺ CD56⁺ CD28⁻ CD27⁻ cells), as well as inaccordance with the present invention within epithelial cells andcytotoxic CD4+ T cells.

Isolated CD160 proteins and polypeptides are available following anyroutine procedure that is available to the skilled person, such as byisolation from CD160-expressing cells, or by recombinant production (seethe above-mentioned reference manuals—Molecular Cloning, A LaboratoryManual; Current Protocols in Molecular Biology).

CD160 protein is of course also available in non-isolated forms, asaccess to a CD160 protein can be achieved through the provision of acell expressing CD160. Cells expressing CD160 as a membrane receptorthus also provide access to a non-isolated form of CD160.

For binding experiments and/or biological activity analysis, cellsexpressing CD160 as a membrane receptor are a preferred source of CD160material. Examples of such cells notably include NK cells and T cellswith cytolytic activity or EC cells or cytotoxic CD4+ T cells collectedfrom human beings, as well as cell lines such as NK92 (ATCC CRL-2407),HUVEC or human microvascular endothelial cells (HMVEC) (Cambrex BioScience, Walkersville, Md.).

CD160 proteins or polypeptides can also be provided in a clustered form.CD 160 proteins or polypeptides can for example be bound to a solidsupport, preferably a biologically-inactive solid support, e.g.CD160-coated beads.

The present invention hence relates to anti-CD160 compounds, and to themedical and/or biological applications thereof.

The anti-CD160 compounds of the invention bind to CD160 substantially onthe same epitope than CL1-R2, and preferably are capable of competingwith the anti-CD160 mAb CL1-R2 (obtainable from the hybridoma depositedas CNCM I-3204) for binding to CD160.

Preferably, the anti-CD160 compounds of the invention are sufficientlyCD160-specific for binding to CD160 without binding to at least one HLAreceptor other than CD160, such as e.g. CD8αβ.

The present invention further relates to a pharmaceutical compositioncomprising at least one anti-CD160 compound of the invention, whereinsaid composition is intended for use in an anti-angiogenic therapy, andnotably to an anti-angiogenic drug.

The present invention also relates to a pharmaceutical compositioncomprising at least one anti-CD160 compound of the invention, whereinsaid composition is intended for the detection of anti-angiogenic sites,and/or for the diagnosis and/or prognosis of a disease or conditioninvolving angiogenesis.

The anti-CD160 compounds of the invention include the anti-CD160 mAbCL1-R2 itself. CL1-R2 has proven very effective in inducing ananti-angiogenic effect upon binding to CD160, whereas prior artanti-CD160 mAb has proven ineffective. It is hence believed thattargeting the correct CD160 epitope on CD160 is crucial to obtain thedesired effect, namely targeting an epitope essentially similar to theone onto which CL1-R2 binds. Hence, the anti-CD160 compounds of theinvention preferably bind to CD160 on an epitope that is essentiallysimilar to the one onto which CL1-R2 binds.

Preferably, the anti-CD160 compounds of the invention bind to humanCD160.

Unspecific binding can induce undesired side effects in the organismreceiving an anti-CD160 compound. More particularly, if a sHLA such assHLA-G were to be administered to a patient in need of ananti-angiogenic effect (for example, a patient having a tumor), saidsHLA would bind to CD160 and induce the desired anti-angiogenic effecton EC, but would also bind to many other receptors expressed by adiversity of different cells within said patient A sHLA such as sHLA-Gwould notably bind to CD8αβ expressed by T cells, and induce apoptosisof these T cells. Such an anti-T effect is highly undesirable to thepatient suffering from a disease such as cancer.

The present invention provides for the first time an anti-CD160 compoundwhich is sufficiently CD160-specific to induce an anti-angiogenesis onEC, without inducing undesired or uncontrolled side effects, such ase.g. apoptosis of T cells.

Hence, the anti-CD160 compounds of the invention preferably do not bindto human CD8αβ.

From this mAb, conservative fragments and derivatives can be easilyproduced by the person of ordinary skill in the art following routineprocedures.

Such conservative fragments and derivatives have retained the desiredbinding affinity and specificity, i.e. they are qualified to be“conservative” because they still bind to substantially the same epitopeas CL1-R2 and/or can compete with CL1-R2 for binding to CD160, and haveretained a sufficient CD160 specificity, such as e.g. a sufficient CD160specificity for not binding to at least one HLA receptor other thanCD160, such as CD8αβ.

According to an advantageous feature of the invention, the anti-CD160compound of the invention does not bind to the T- and NK-expressedreceptor CD85j (also referred to as ILT-2). Preferably, they do not bindto human CD85j.

Preferably, the anti-CD160 compound of the invention does notcross-react with any EC receptor other than CD160.

Most preferably, the anti-CD160 compounds of the invention are fullyCD160-specific, in the sense that they do not cross-react with anyclassical and non classical HLA molecule receptor with either allele orbroad specificity. These receptors include CD8αβ, CD94 associated witheach of the NKG2 family gene products (located on chromosome 12), andall the products of the genes located on chromosome 19 including KIR andILT/LIR families.

Binding or absence of binding of an anti-CD160 compound of the inventionto a receptor is meant as binding or absence of binding as would beobserved under physiological conditions, or under in vitro conditionsmimicking in vivo conditions. Any mean and/or procedure that the skilledperson would find appropriate to perform said binding assay is suitablefor determining whether a compound binds to CD160, does not bind to anyother EC receptor, does not bind to CD8αβ, and does not bind to CD85j.

Illustrative conditions comprise providing a cell expressing the desiredtarget, such as an EC (expressing CD160 and other EC receptors), or aCD8+ T (expressing CD160 and CD8αβ), or as will be shown below CD4+ Tcells (expressing CD160 as demonstrated by the present invention), andcontacting said CD160-expressing cell with the compound under conditionsof compound concentration, contact duration, pH, and temperature thatwould enable binding of the cell-expressed target by its natural ligand.

Illustrative techniques to assess whether a compound binds to CD160 butnot to at least one other HLA receptor, such as CD8αβ notably comprise:

-   -   flow cytometry analyses with transfected cells expressing each        of the gene products capable to bind HLA molecules (CD160,        CD8αβ, etc.), and/or    -   sensor chips (such as the sensor chips BR-1000-14, BIACORE AB,        Uppsala, Sweden), which can be coated by soluble recombinant HLA        ligands such as CD160, CD8αβ, etc. using a Biacore (BIACORE AB,        Uppsala, Sweden).

Sources of CD160-expressing cells comprise EC collected from a healthyindividual, or EC from a cell line such as NK92 (ATCC Number CRL-2407),HUVEC, HMVEC (Cambrex Bio Science, Walkersville, Md., U.S.A.).

Sources of CD8αβ-expressing cells comprise CD8+ T cells, such as CD8+ Tcells collected from a healthy individual, or CD8+ T cells from a cellline such as MOLT-4 (ATCC Number CRL-1582).

Sources of CD85j-expressing cells comprise CD85j+ T cells, such asCD85j+ T cells or monocytes collected from a healthy individual, orCD85j+ T cells from a cell line such as NAMALWA (ATCC Number CRL-1432).

Preferred sources are those which express the human form of the targetreceptor.

The human sequence of CD160 is available from the NCBI data bank underaccession numbers NM_(—)007053 (nucleic acid) and CAG46686 (protein) Thehuman sequence of CD8α is available from the NCBI data bank underaccession numbers M27161 (nucleic acid) and AAA59674(protein). The humansequence of CD8β is available from the NCBI data bank under accessionnumbers M36712 (nucleic acid) and AAA35664 (protein).

The human sequence of CD85j is available from the NCBI data bank underaccession numbers BC015731 and NM_(—)006669 (nucleic acid) and AAH15731and NP_(—)006660.1(protein).

The anti-CD160 compounds of the invention are anti-angiogenic agents,and are thereby useful for preventing or treating a tumor, such as acarcinoma, or a leukaemia (e.g. B-cell chronic lymphocytic leukaemia).

They are also useful for preventing or treating pre-eclampsia oreclampsia, and/or for preventing or treating diabetes, an ischemicocular disease, or rheumatoid arthritis.

An illustrative anti-CD160 compound of the invention comprises saidCL1-R2 mAb. From this mAb conservative fragments and derivatives can beproduced by the skilled person following routine procedures. Suchconservative fragments and derivatives are functional equivalents ofsaid CL1-R2 mAb.

An “antibody fragment” is a portion of an antibody such as a heavychain, a light chain, a VH, a VL, Fab, a Fab′, a F(ab)2, a F(ab′)2, andthe like, as well as each minimal recognition units consisting of theamino acid residues that mimic the hypervariable region (CDR1H, CDR2H,CDR3H, CDR1L, CDR2L, CDR3L). Such fragments are obtainable by routineprocedures, such as proteolytic digestion (for example, pepsin digestionto generate F(ab′)2 ; papain digestion to generate Fab).

Preferred fragments of the invention are those which are conservative,i.e. those CL1-R2 fragments which have retained said desired CD160binding affinity and specificity (i.e. have retained the feature ofbinding to CD160 on substantially the same epitope as CL1-R2 and/orcapable of competing with CL1-R2 for binding to CD160, and the featureof binding to CD160 without binding to at least one HLA receptor otherthan CD160, such as e.g. CD8αβ). Preferred conservative fragment of mAbCL1-R2 comprise Fab, Fab′, F(ab)2, F(ab′)2 or Fv fragments of said mAbCL1-R2.

Such conservative fragments may be used as such, for biological and/ormedical applications.

Non conservative fragments such as a CL1-R2 CDR in isolated form arenevertheless also an object of the present invention, as they can becombined together to form a conservative derivative of CL1-R2.

The anti-CD160 compounds of the invention also comprise the conservativederivatives of said mAb CL1-R2, i.e. any anti-CD160 compound:

-   -   which is a CL1-R2 derivative in the sense that it comprises at        least one CL1-R2 fragment (preferably at least one CDR of        CL1-R2, preferably at least one CDR3 of CL1-R2), and    -   which is also conservative in the sense that the resulting        derivative has retained an affinity for binding to CD160, and        has also retained said CD160 binding specificity (i.e. have        retained the feature of binding to CD160 on substantially the        same epitope as CL1-R2 and/or capable of competing with CL1-R2        for binding to CD160, and the feature of binding to CD160        without binding to at least one HLA receptor other than CD160,        such as e.g. CD8αβ).

From CL1-R2, conservative derivatives are indeed obtainable by theskilled person through synthesis and/or genetic engineering.

Illustrative conservative derivatives are obtainable by the skilledperson pursuant to our routine procedures.

The conservative derivative of the invention may be monovalent (oneCD160 binding site), or multivalent (at least two CD160 binding sites).Preferred multivalent conservative derivatives include tetravalentconservative derivatives.

They e.g. include derivatives which are chimaeric antibodies obtainableby grafting at least one Fv fragment of CL1-R2 to an Fe fragment derivedfrom another antibody. The Fe fragment is preferably chosen to be asless immunoreactive as possible for the organism to which said drug isto be administered. For example, when the drug is intended foradministration to a human being, said Fc fragment preferably is a humanFe fragment. Conservative derivatives of the invention also includehumanized antibodies, obtainable by grafting at least one CL1-R2 CDRonto a human antibody frame region (hFR). The objective is here also toprovide the organism into which said drug is to be administered with acompound that induces as few as undesired immunogenic side effects aspossible. Conservative derivatives of the invention also includederivatives obtainable by grafting at least one CL1-R2 VH region to atleast one VL region, optionally via a linker (L), such as a peptidelinker. Such molecules are known to the skilled person as scFv. They canbe monomeric or multimeric. Appropriate linkers are those which allowthe VH and VL domains to fold into a single polypeptide chain which hasa three dimensional structure very similar to the original structure ofthe whole antibody, and thus maintain the binding specificity. Suchappropriate linkers are known to the skilled person. An illustrativemethod to produce such linkers is described in WO 88/01649 in the nameof GENEX Corp. (U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,260,203).

Said conservative derivative of mAb CL1-R2 may be monovalent, ormultivalent.

An illustrative conservative derivative of mAb CL1-R2 comprises at leastone scFv compound comprising at least one CL1-R2 VH region of CL1-R2linked to at least one CL1-R2 VL region of CL1-R2 via a peptide linker(L).

The scFv can have a VL-L-VH orientation (see e.g. WO 88/01649 in thename of GENEX Corp.—U.S. Pat. No. 4,946,778 and U.S. Pat. No.5,260,203—), or a VH-L-VL orientation (see e.g. WO 88/09344 in the nameof CREATIVE BIOMOLECULES Inc.—U.S. Pat. No. 5,132,405; U.S. Pat. No.5,091,513; U.S. Pat. No. 5,258,498; U.S. Pat. No. 5,476,786; U.S. Pat.No. 5,482,858;U.S. Pat. No. 6,207,804 B1—).

The scFv can be monovalent or multivalent (aggregation of several scFv).

Illustrative conservative derivative notably comprises a scFv multimerderived from said CL1-R2 mAb, joined to a Fc fragment.

Another illustrative conservative derivative of mAb CL1-R2 is a compoundcomprising at least one Fv fragment of CL1-R2 linked to a human Fc.

Another illustrative conservative derivative of mAb CL1-R2 is obtainableby adding one or more Fab derived from said CL1-R2 mAb at the C-terminusof each H chain of the full length CL1-R2 mAb.

Another illustrative conservative derivative of mAb CL1-R2 is obtainableby covalently linking full-length CL1-R2 mAbs together to form anaggregated Ab form.

Another illustrative conservative derivative of mAb CL1-R2 is obtainableby linking two or more Fabs head-to-tail.

A multivalent scFv according to the present invention is obtainable bylinking at least two scFv in a multimer. Linking can be achievedcovalently or non-covalently. Illustrative multivalent scFv aretetrameric scFv. Multivalent scFv have more than one binding site.Hence, multimeric scFv having several CD160 binding sites can beproduced, to provide an anti-CD160 compound with enhanced avidity forCD160. Such multimeric scFv are particularly advantageous according tothe present invention.

Multimeric scFv can be monospecific, i.e. all of their binding sitestarget CD160.

Alternatively, multimeric scFv can comprise one or more CD160 bindingsite(s), as well as one or more other binding site(s) for binding to atarget different from CD160. Such other binding site(s) may e.g. targeta compound that is different from CD160, but still expressed by EC, soas to direct the action of the compound towards EC more efficiently.

Examples of such other target(s) comprise VEGF receptors and allreceptors that induce EC growth upon ligation with their physiologicalligand.

Bi- or multi-specific multimeric scFv are particularly advantageoustherapeutic means. Methods to produce multimeric scFv are known to theskilled person, see e.g. WO 94/13806 in the name of The DOW CHEMICALCompany (U.S. Pat. No. 5,877,291 and U.S. Pat. No. 5,892,020), WO93/11161 in the name of ENZON Inc. (U.S. Pat. No. 6,515,110 B1; U.S.Pat. No. 6,121,424; U.S. Pat. No. 6,027,725;U.S. Pat. No. 5,869,620).

Other conservative multivalent derivatives are obtainable by:

-   -   joining a scFv multimer to a Fc fragment, or by    -   adding one or more Fab at the C-terminus of each H chain of the        full length CL1-R2 IgG, or by    -   covalently linking full-length CL1-R2 to form multivalent Ab, or        by    -   linking two or more Fabs head-to-tail (see e.g. Miller et al.        2003 “Design, Construction, and in vitro analysis of multivalent        antibodies” The Journal of Immunology, 170:4854-4861).

The present invention provides a pharmaceutical composition comprisingas an active ingredient an anti-CD160 compound of the invention, for usein diagnosis and/or prognosis and/or therapy.

Said composition may be in any pharmaceutical form suitable foradministration to a patient, including but not limited to solutions,suspensions, lyophilized powders, capsule and tablets. Thepharmaceutical compositions of the invention may further comprise anypharmaceutically acceptable diluent, carrier, excipient or auxiliary.

The pharmaceutical composition of the invention may be formulated forinjection, e.g. local injection, transmucosal administration,inhalation, oral administration and more generally any formulation thatthe skilled person finds appropriate to achieve the desired prognosisand/or diagnosis and/or therapy.

The anti-CD160 compound of the invention is contained in saidpharmaceutical composition in an amount effective to achieve theintended purpose, and in dosages suitable for the chosen route ofadministration. More specifically, a therapeutically effective dosemeans an amount of a compound effective to prevent, alleviate orameliorate symptoms of the disease or condition of the subject beingtreated, or to arrest said disease or condition.

Depending on the intended application, the anti-CD160 compounds of theinvention, whether as CL1-R2 fragments or as CL1-R2 derivatives, mayfurther comprise additional constituents.

For example, when the anti-CD160 compound of the invention is intendedfor prognosis or diagnosis, it may further comprise a detectable label,such as a fluorochrom, or an entity with enzymatic activity, or withradioactivity, and more generally any entity enabling the detection ofsaid compound.

When the compound is intended for therapeutic administration to anorganism in need thereof, it may further comprise an immunotoxin and/ora radioelement.

The anti-CD160 compounds of the invention may of course alternatively beused for the detection of anti-angiogenic sites. The present inventionhence also relates to a pharmaceutical composition or kit comprising atleast one anti-CD160 compound of the invention, which is intended forthe detection of anti-angiogenic sites.

The present invention also relates to the use of an anti-CD160 compoundof the invention, for the identification of an anti-angiogenic compound.

The present invention indeed provides the demonstration that CD160 isexpressed by endothelial cells (EC), and that the anti-CD160 compoundsof the invention bind to EC-expressed CD160 and thereupon induce ananti-angiogenic effect on said EC.

The anti-CD160 compounds of the invention have the advantageous abilityto act as an activating extracellular ligand of CD160.

Equivalents compounds can hence be found by isolation and/oridentification of compounds that show equivalent affinity andspecificity for binding to CD160, i.e. that have the ability to competewith an anti-CD160 compound of the invention (such as CL1-R2 itself) forbinding to CD160, and that are sufficiently CD160-specific for bindingto CD160 without binding to at least one HLA receptor other than CD160,such as CD8αβ.

Such an identification and/or isolation can be achieved by e.g.screening method, such as e.g. high throughput screening.

The present invention hence also relates to a pharmaceutical compositionor a kit comprising at least one anti-CD160 compound of the invention,said pharmaceutical composition or kit being intended for theidentification and/or isolation of an anti-angiogenic compound.

The present invention thus also relates to the use and more particularlythe in vitro use of the anti-CD160 mAb CL1-R2 (obtainable from thehybridoma deposited as CNCM I-3204), or of a conservative fragmentthereof, or of a conservative derivative thereof, for the identificationand/or isolation of an anti-angiogenic compound, wherein said fragmentor derivative is capable of competing with CL1-R2 for binding to CD160,and is sufficiently CD160-specific for binding to CD160 without bindingto at least one HLA receptor other than CD160, such as CD8αβ, andwherein said derivative comprises at least one CL1-R2 fragment.

More particularly, the present invention encompasses a method toidentify an anti-angiogenic compound, characterized in that itcomprises:

-   -   providing a candidate compound,    -   determining whether said candidate compound:        -   has the ability to compete with CL-R2 for binding to CD160,            and        -   does not bind to at least one HLA receptor other than CD160,            such as CD8αβ,    -   identifying said candidate compound as being an anti-angiogenic        specific compound if it actually has said CD160 binding affinity        and specificity.

Any candidate compound that the skilled person finds appropriate may beprovided for implementation of the method of the invention. Illustrativecandidate compounds may e.g. be found in chemical or biologicalcollections, such as e.g. viral peptides or peptides deriving frompathogens (for example Cytomegalovirus peptides).

The CD160 target to be used for implementation of the methods of theinvention may be provided in any form that the skilled person findsappropriate. It may e.g. be provided in the form of a cell expressingCD160 as a functional membrane receptor. Illustrative cells notablycomprise EC. EC are obtainable from cell lines such as HUVEC, HMVEC,NK92 (see example 2 below). EC are also obtainable by collection andisolation from a healthy individual. The CD160 target may also beprovided in the form of soluble recombinant CD160 proteins (Flag-CD160or GST-CD160).

The present invention also relates to the use, and more particularly thein vitro use of an anti-CD160 compound of the invention as a CD160activating ligand to identify a CD160 molecular effector or transducer,i.e. the use of an anti-CD160 compound of the invention as a CD160ligand to identify a molecule which is involved in the anti-angiogenicsignal transduction mediated by an EC-expressed CD160.

Such effectors and transducers are preferred cell targets foranti-angiogenic drugs, such as anti-tumor drugs.

The present invention hence also relates to a pharmaceutical compositionor a kit comprising at least one anti-CD160 compound of the invention,said pharmaceutical composition or kit being intended for theidentification and/or isolation of lipid-RAFT associated membranemolecule that is involved in CD160 anti-angiogenic signal transduction,and/or of a secondary messenger that is involved in CD160anti-angiogenic signal transduction.

The present invention also relates to a method to identify a lipidRAFT-associated membrane molecule which is involved in CD160anti-angiogenic signaling pathway when expressed by an endothelial cell,characterized in that it comprises:

-   -   activating a CD160 expressed on an EC with CL1-R2 or with a        conservative fragment or derivative thereof, e.g. by providing a        CD160-expressing EC and contacting it with CL1-R2 or with a        conservative fragment or derivative thereof so as to aggregate        CD160,    -   lysing said cell so as to recover the lipid RAFT domain fraction        of said cell, e.g. by lysing said cell so as to dissociate the        membrane complexes (e.g. by using a strong detergent such as        NP40), and recovering a fraction of said lysate comprising at        least one lipid RAFT domain),    -   identifying within said RAFT fraction at least one compound        which appear CD160-specific:        -   by comparison with those control compounds which are            obtained under similar conditions but using a non-reactive            isotype-matched control Ab instead of said CL1-R2 or            conservative fragment or derivative, and        -   by comparison with those control compounds which are            obtained under similar conditions but using a compound which            does not bind to CD160 but binds to another EC-expressed            receptor,    -   optionally, recovering said at least one CD160-specific compound        thus identified,    -   optionally, sequencing or micro-sequencing this(these)        compound(s).

whereby said at least one CD160-specific compound thus identified is alipid RAFT-associated membrane molecule that is involved in CD160anti-angiogenic signaling pathway.

To achieve the required comparison with said controls, any mean and/ormethod that the skilled person may find appropriate to compare proteinpatterns can be used.

For example, said RAFT fraction may e.g. be placed for migration in a2-dimension gel (pH/PM), and the protein spots revealed with silvernitrate.

Said non-reactive isotype-matched control Ab is a non-relevant Ab whichhas the same isotype as CL1-R2, but which does not bind to CD160, anddoes also not bind to any compound that may be found within or on saidEC. Said non-reactive isotype-matched control Ab may e.g. be anon-relevant mouse Ig.

Said compound which does not bind to CD160 but binds to anotherEC-expressed receptor may e.g. be an Ab directed to an EC receptor otherthan CD160, such as an anti-VEGF receptor when EC is used.

Any CD160-expressing EC may be used. Illustrative cells notably compriseEC obtainable from cell lines such as HUVEC, HMVEC, NK92 (see example 2below), or by collection and isolation from a healthy individual.

The present invention also relates to a method to identify a secondarymessenger which is involved in CD160 anti-angiogenic signal transductionwhen expressed by an endothelia cell, characterized in that itcomprises:

-   -   activating a CD160 expressed on an EC with CL1-R2, e.g. by        providing a CD160-expressing EC and contacting it with CL1-R2 so        as to aggregate CD160,    -   lysing said cell under mild conditions so as to essentially        preserve the putative complexes formed on CD160 (e.g. by using a        mild detergent such as BRIJ58® or BRIJ98®—SIGMA—),    -   optionally pre-clearing the lysate,    -   recovering CL1-R2 as well as any compound that may be associated        thereto, e.g. by immunoprecipitation with a goat anti-mouse Ab,    -   achieving an in vitro kinase assay,    -   identifying at least one compound which has incorporated at        least one phosphorus compound as a result of said in vitro        kinase assay,

whereby said at least one identified compound is a secondary messengerthat is involved in CD160 (anti-angiogenic) signal transduction,

-   -   optionally recovering said at least one identified compound,    -   when said at least one identified compound comprises a protein        or a polypeptide constituent:        -   optionally achieving a trypsin digestion of said at least            one recovered compound,        -   optionally sequencing or microsequencing said at least one            recovered compound and comparing the peptide sequence thus            obtained with those available on protein data banks, or            following a mass spectrometry procedure such the one            described by Bruyns E, Marie-Cardine A, Kirchgessner H,            Sagolla K, Shevchenko A, Mann M, Autschbach F, Bensussan A,            Meuer S, Schraven B. <<T cell receptor (TCR) interacting            molecule (TRIM), a novel disulfide-linked diner associated            with the TCR-CD3-zeta complex, recruits intracellular            signalling proteins to the plasma membrane>> J Exp Med. 1998            Aug. 3;188(3):561-75.,    -   so as to obtain the sequence of said protein or polypeptide        constituent.

In vitro kinase assays are well-known to the skilled person. Adetectable phosphorus compound (such as radioactive phosphorus providedby e.g. P³²-ATP, a fluorescent or a luminescent phosphorus compound) isusually used for such in vitro kinase assay. An illustrativeexperimental procedure is described in Bruyns E, Marie-Cardine A,Kirchgessner H, Sagolla K, Shevchenko A, Mann M, Autschbach F, BensussanA, Meuer S, Schraven B. <<T cell receptor (TCR) interacting molecule(TRIM), a novel disulfide-linked dimer associated with the TCR-CD3-zetacomplex, recruits intracellular signalling proteins to the plasmamembrane>> J Exp Med. 1998 Aug. 3;188(3):561-75.

To identify said at least one compound which has incorporated at leastone phosphorus compound, any mean and/or procedure that the skilledperson finds appropriate may be used. It may e.g. be proceeded bymigration of said fraction of CL1-R2 complex on a polyacrylamide gel,optionally western blotting with anti-phosphoTyr and/or phosphoSerand/or phosphoThr, and detecting incorporated phosphorylation (with aradioactivity scintillation counter when P³² has been used), recoveringthe corresponding band (e.g. by elution).

An illustrative experimental procedure can also be found by the skilledperson also in Nikolova et al. 2002 (“BY55/CD160 acts as a co-receptorin TCR signal transduction of a human circulating cytotoxic effector Tlymphocyte subset lacking CD28 expression” International Immunology vol.14, No. 5, p. 445-451).

Illustrative secondary messengers that are involved in CD160(anti-angiogenic) signal transduction have been identified by theinventors. They notably comprise pi-3-kinase and lck (p56).

Inhibitors of membrane-associated molecules and/or of cytosolic secondmessenger may have therapeutic applicability. They may advantageously beassociated with a compound increasing the specificity of their delivery.

NK and T Cells and the Immune System:

The present invention provides the demonstration that cytokineproduction by NK and T cells uses the CD160 signaling pathway in NK andT cells, and that it can be controlled by aggregated anti-CD160compounds for up-regulation, or by soluble anti-CD160 compounds orCD160-CD158b cross-linking agents for down-regulation.

The present invention provides with anti-CD160 specific compounds thatcan specifically exert these controls on CD160.

The present invention also demonstrates that cross-linking CD160 toCD158b induces an inhibition of CD160 activation, thereby resulting inan inhibition of the cytokine production.

The cytokine profile that is induced by stimulation of CD160 is uniquecompared to the one obtained by stimulation of other NK-expressedreceptors such as CD 16 or NKG2D. The CD160-triggered cytokine profileis unique also in the sense that it very closely mimics the one obtainedby stimulation with the natural CD160 ligand (sHLA).

Stimulation of CD160 induces the production and secretion of IFNγ, TNFαand IL-6. Except for the natural ligand sHLA, it is the first that timethat there is provided a ligand that induces IL-6 production from NKcells.

The CD160 ligands provided by the present invention are anti-CD160specific compounds. They notably comprise the anti-CD160 monoclonalantibody referred to by the inventors as CL1-R2. A CL1-R2 producinghybridoma has been deposited within the Collection Nationale de Culturesde Microorganismes in accordance with the Budapest Treaty under CNCMdeposit accession number I-3204 (C.N.C.M. Institut Pasteur 25, rue duDocteur Roux F-75724 Paris Cedex 15 France).

The present invention also describes that CD160 is expressed by CD4+ Tcells. Such CD160 detections could and can be made because the presentinvention provides a publicly-available anti-CD160 specific compound.CD160+ CD4+ cells have notably been identified within a skin sample froma human patient suffering from atopic dermatitis.

The anti-CD160 compounds of the invention which are useful forregulating NK and T cells cytokine production are identical to thosewhich have been above-described for EC and angiogenesis: they comprisethe mAb CL1-R2 of the invention as well as the conservative fragmentsand derivatives thereof. The structural description, the affinity andspecificity properties that have been described for the anti-CD160compounds of the invention in the context of EC angiogenesis hence applymutatis mutandis to the anti-CD160 compounds of the invention in thecontext of regulation of NK and T cell cytokine production.

Also, similarly to what has been described in detail in the context ofEC angiogenesis regulation, unspecific binding or binding to undesiredtargets, i.e. HLA receptors other than CD160, such as CD8αβ and/or CD85jand/or CD4 is not advantageous, as such compounds would induceuncontrolled chain reaction in the organism to which they would beadministered. They would notably induce T cell apoptosis if they werecomprising an anti-CD8 ligand.

There however is a functional difference between the anti-CD160compounds of the invention when used as ligands of CD160 expressed as animmune receptor on NK and/or T cells, and the anti-CD160 compounds ofthe invention when used as ligands of CD160 expressed as an endothelialcell receptor.

When it relates to NK and T cells and cytokine production, it shouldindeed be functionally discriminated between soluble and aggregatedanti-CD160 compounds.

The soluble anti-CD160 compounds of the invention induce an inhibitionof CD160 signalling pathway (i.e. inhibition of cytokine production),whereas the aggregated forms of the anti-CD160 compounds of theinvention induces a CD160 stimulation (i.e. induction of, or stimulationof cytokine production).

The present invention hence also relates to anti-CD160 compounds whichcomprise with the anti-CD160 mAb CL1-R2 (obtainable from the hybridomadeposited as CNCM I-3204), and any compound which is capable ofcompeting with CL1-R2 for binding to CD160, and which is sufficientlyCD160-specific for binding to CD160 without binding to at least one HLAreceptor other than CD160, such as and preferably CD8αβ.

Preferably, the anti-CD160 compounds of the invention do further notbind to CD85j and/or CD4.

Most preferably, the anti-CD160 compounds of the invention do not bindto any HLA receptor other than CD160.

The present invention also relates to a pharmaceutical composition, suchas a drug, comprising an anti-CD160 compound of the invention.

Such a drug is useful for inducing or inhibiting, and/or up- ordown-regulating the cytokine production of an individual. Said cytokinesnotably comprise IFNγ and/or TNFα and/or IL-6.

Such a drug is useful for the (curing and/or preventing and/orpalliative) treatment of any disease or condition involving an excessiveor an insufficient cytokine production.

Such a drug can thus be useful for inducing or inhibiting, and/or up- ordown-regulating the adaptive immunity potential of said individual. Itthus enables the regulation of a Th1 response.

Said drug may also be intended for the treatment or prevention of aninfection.

Said drug may also be intended as an additional product, such as anadjuvant, in a vaccine procedure to induce and/or amplify specificcytotoxic T lymphocyte (CTL) responses.

Said drug may also be intended for inducing or inhibiting, and/or up- ordown-regulating hematopoiesis in an individual, for the (curing orpalliative or preventive) treatment of irradiated individuals and/or forthe treatment or prevention of bone marrow aplasia. Such a drug wouldthen be very useful to patients that have been submitted to irradiationin a pre-graft treatment or as an anti-tumor treatment: the anti-CD160compounds of the invention can indeed help them in restoring their bloodcell population.

Said drug may also be intended for inducing or inhibiting, and/or up- ordown-regulating an inflammatory reaction in said individual, and/or forthe treatment or prevention of an allergy in said individual, such asatopic dermatitis.

Said drug may also be intended to induce a vasodilatation.

When it is intended for inhibiting and/or down-regulating the cytokineproduction of an individual, an anti-CD160 compound of the invention maycomprise at least one CD158b binding site in addition to its CD160binding site(s). Cross-linking of CD160 and CD158b indeed induces aninhibition of CD160 signaling pathway.

Alternatively, the anti-CD160 of the invention may be provided insoluble form. When provided in soluble form, the anti-CD160 compounds ofthe invention indeed inhibit CD160 signaling pathway. By “soluble” form,it is herein meant a “soluble” form as intended by the skilled person inthe field of immune system receptor-ligand interactions. Moreparticularly, the fact that a ligand is in soluble form implies thatsaid ligand has one or two, but no more than two, binding site(s) forthe activating target, i.e. in the present for CD160.

Conversely, the fact that a ligand is in aggregated form implies thatsaid ligand has at least two binding sites for the activating target,i.e. in the present for CD160.

The anti-CD160 compounds of the invention in soluble form comprise theanti-CD160 mAb obtainable from hybridoma CNCM I-3204 (IgG).

They also comprise the conservative fragments of CL1-R2, i.e. the CL1-R2fragments that have retained an affinity for binding to CD160, and moreparticularly the ability to compete with CL1-R2 for binding to CD160,and that have retained a sufficient CD160-specificity for binding toCD160, without binding to at least CD8αβ. Such conservative fragmentsnotably comprise the Fab, Fab′, F(ab)2, F(ab′)2 and Fv fragments of saidmAb CL1-R2.

The anti-CD160 compounds of the invention in soluble form also comprisemono- or divalent conservative derivatives of CL1-R2, i.e. a compound:

-   -   which comprises at least one fragment of said CL1-R2 mAb, and    -   which has retained an ability to compete with CL1-R2 for binding        to CD160, and has also retained a sufficient CD160 specificity        for binding to CD160 without binding to at least CD8αβ,    -   wherein said derivative has one or two CD160 binding site(s).

Illustrative mono- or divalent conservative derivatives of CL1-R2comprise:

-   -   any humanized Ab form of CL1-R2, or    -   any chimaeric Ab form of CL1-R2, or    -   any mono- or divalent scFv derived from CL1-R2, optionally        joined to a Fc fragment, or    -   a CL1-R2 Fv fragment linked to a Fc fragment, or    -   a full length CL1-R2 Ab comprising one additional CL1-R2 Fab at        each its H chain.

When it is intended for inducing and/or up-regulating the cytokineproduction of an individual, the anti-CD160 compounds of the inventionmay be provided in an aggregated form, i.e. as a compound comprising atleast three CD160 binding sites and no CD158b binding site.

Such aggregated forms of the anti-CD160 compounds of the invention areobtainable by aggregation of the soluble forms of the anti-CD160compounds of the invention. Aggregated forms can be obtained by geneticmanipulation or chemically with linkers.

The present invention also relates to the use of the anti-CD160 mAbCL1-R2.(obtainable from the hybridoma TM60 deposited as CNCM I-3204), orof a conservative fragment thereof, or of a conservative derivativethereof, for the identification and/or isolation of a compound havingthe ability to induce or inhibit, and/or to up- or down-regulate thecytokine production of a NK and/or a T CD8+ and/or a T CD4+ cell,

wherein said fragment or derivative is capable of competing with CL1-R2for binding to CD160, and is sufficiently CD160-specific for binding toCD160 without binding to at least CD8αβ, and

wherein said derivative comprises at least one CL1-R2 fragment.

More particularly, the present invention encompasses a method toidentify a compound having the ability to induce or inhibit, and/or todown- or up-regulate the cytokine production of a NK cell and/or a TCD8+ cell and/or a T CD4+ cell, characterized in that it comprises:

-   -   providing a candidate compound,    -   determining whether said candidate compound:        -   has the ability to compete with CL1-R2 for binding to CD160,            and        -   does not bind to at least CD8αβ,    -   identifying said candidate compound as having the ability to        induce or inhibit, and/or to down- or up-regulate the cytokine        production of a NK cell and/or a T CD8+ cell and/or a T CD4+        cell compound if it actually has said CD160 binding affinity and        specificity.

The present invention also encompasses the use of the anti-CD160 mAbCL1-R2 (obtainable from the hybridoma deposited as CNCM I-3204), or of aconservative fragment thereof, or of a conservative derivative thereof,as a CD160 ligand to identify a molecule which is involved in theCD160-mediated cytokine production of a NK cell and/or a T CD8+ celland/or a T CD4+ cell,

wherein said fragment or derivative is capable of competing with CL1-R2for binding to CD160, and is sufficiently CD160-specific for binding toCD160 without binding to at least CD8αβ, and

wherein said derivative comprises at least one CL1-R2 fragment.

More particularly, the present invention relates to a method to identifya molecule which is involved in the CD160-mediated cytokine productionof a NK cell and/or a T CD8+ cell and/or a T CD4+ cell, and which isexpressed by said cell as a lipid RAPT-associated membrane molecule,characterized in that it comprises:

-   -   activating a CD160 expressed by a NK cell and/or a T CD8+ cell        and/or a T CD4+ cell, with CL1-R2 or with a conservative        fragment or derivative thereof, e.g. by providing a        CD160-expressing NK cell and/or a T CD8+ cell and/or a T CD4+        cell, and contacting it with CL1-R2 or with a conservative        fragment or derivative thereof so as to aggregate CD160,    -   lysing said cell so as to recover the lipid RAFT domain fraction        of said cell, e.g. by lysing said cell so as to dissociate the        membrane complexes (e.g. by using a strong detergent such as        NP40), and recovering a fraction of said lysate comprising at        least one lipid RAFT domain),    -   identifying within said RAFT fraction at least one compound        which appear CD160-specific:        -   by comparison with those control compounds which are            obtained under similar conditions but using a non-reactive            isotype-matched control Ab instead of said CL1-R2 or            conservative fragment or derivative, and        -   by comparison with those control compounds which are            obtained under similar conditions but using a compound which            does not bind to CD160 but binds to another receptor            expressed by said cell, (such as anti-CD16 or an anti-CD2            antibody when a NK cell is used, an anti-CD8 antibody when a            T CD8+ cell is used, an anti-CD4 antibody when a T CD4+ cell            is used),    -   optionally, recovering said at least one CD160-specific compound        thus identified,    -   optionally, sequencing or micro-sequencing this(these)        compound(s),

whereby said at least one CD160-specific compound thus identified is amolecule which is involved in the CD160-mediated cytokine production ofsaid cell, and which is expressed by said cell as a lipidRAFT-associated membrane molecule.

The anti-CD160 compounds of the invention also allows for theidentification of those molecules which are expressed in the cytoliccompartment of an NK cell and which are involved in the CD160 signaltransduction, thereby mediated an up- or down-regulation of the cytokineproduction of said NK cell.

The present invention hence also relates to a method to identify asecondary messenger which is involved in the CD160-mediated cytokineproduction of a NK cell and/or a T CD8+ cell and/or a T CD4+ cell,characterized in that it comprises:

-   -   activating a CD160 expressed on a NK cell and/or a T CD8+ cell        and/or a T CD4+ cell, with CL1-R2, e.g. by providing a        CD160-expressing NK (i.e. a cytotoxic NK and/or a T CD8+ cell        and/or a T CD4+ cell and contacting it with CL1-R2 so as to        aggregate CD160,    -   lysing said cell under mild conditions so as to essentially        preserve the putative complexes formed on CD160 (e.g. by using a        mild detergent such as BRIJ58® or BRIJ98®—SIGMA—),    -   optionally pre-clearing the lysate,    -   recovering CL1-R2 as well as any compound that may be associated        thereto, e.g. by immunoprecipitation with a goat anti-mouse Ab,    -   achieving an in vitro kinase assay,    -   identifying at least one compound which has incorporated at        least one phosphorus compound as a result of said in vitro        kinase assay,

whereby said at least one identified compound is a secondary messengerwhich is involved in the CD160-mediated cytokine production of saidcell,

-   -   optionally recovering said at least one identified compound, and    -   when said at least one identified compound comprises a protein        or a polypeptide constituent:        -   optionally achieving a trypsin digestion of said at least            one recovered compound,        -   optionally sequencing or microsequencing said at least one            recovered compound and comparing the peptide sequence thus            obtained with those available on protein data banks, or            following a mass spectrometry procedure such the one            described by Bruyns E, Marie-Cardine A, Kirchgessner H,            Sagolla K, Shevchenko A, Mann M, Autschbach F, Bensussan A,            Meuer S, Schraven B. <<T cell receptor (TCR) interacting            molecule (TRIM), a novel disulfide-linked dimer associated            with the TCR-CD3-zeta complex, recruits intracellular            signalling proteins to the plasma membrane>> J Exp Med. 1998            Aug. 3;188(3):561-75.,    -   so as to obtain the sequence of said protein or polypeptide        constituent.

As above-mentioned, in vitro kinase assay are well-known to the skilledperson. A detectable phosphorus compound (such as radioactive phosphorusprovided by e.g. P³²-ATP, a fluorescent or a luminescent phosphoruscompound) is usually used for such in vitro kinase assay.

An illustrative experimental procedure is described in Bruyns E,Marie-Cardine A, Kirchgessner H, Sagolla K, Shevchenko A, Mann M,Autschbach F, Bensussan A, Meuer S, Schraven B. <<T cell receptor (TCR)interacting molecule (TRIM), a novel disulfide-linked dimer associatedwith the TCR-CD3-zeta complex, recruits intracellular signallingproteins to the plasma membrane>> J Exp Med. 1998 Aug. 3;188(3):561-75.

An illustrative experimental procedure can also be found in Nikolova etal. 2002 (“BY55/CD160 acts as a co-receptor in TCR signal transductionof a human circulating cytotoxic effector T lymphocyte subset lackingCD28 expression” International Immunology vol. 14, No. 5, p. 445-451).

Illustrative secondary messengers that are involved in CD160(anti-angiogenic) signal transduction have been identified by theinventors. They notably comprise pi-3-kinase and lck (p56).

The present invention also relates to:

-   -   the use of a T CD4+ cell as a source of, or as a provider of        CD160 receptor,    -   the use of CD160 as CD4 co-receptor, and    -   the use of CD160 as a receptor to induce or stimulate cytokine        production by a NK cell, and/or by a T CD8+ cell, and/or a T        CD4+ cell,    -   the use of a NK cell as an IL-6 producer.

The present invention also encompasses the use of an anti-CD160 compoundof the invention to induce CTL differentiation.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C. HLA-C triggers cytokine production by NK92 and PB-NKcells.

(FIG. 1A) IL-2 treated NK92 cells were co-cultured for 4 h withK562_(Class-I+) in the absence or presence of blocking concentrations ofW6/32 anti-HLA-C mAb (lower panel). Cells were fixed, permeabilized, andstained for intracellular TNF-α expression, as described in Materialsand Methods. K562_(Class-I+) or NK92 cells alone were used as controls(upper panel).

(FIG. 1B) K562_(Class-I+), K562, and K562-Cw5 were analyzed by flowcytometry for surface expression of HLA-C using W6/32 mAb, followed byPE-conjugate (open profiles). Dark profiles are Ig-isotype controlstaining.

(FIG. 1C) Simultaneous measurement of IL-4, IL-6, IL-10, TNF-α and IFN-γproduction by PB-NK after 16 h of culture alone or co-culture withK562_(Class-I+), K562 or K562-Cw5.

Supernatants were collected for CBA analysis. The samples were acquiredusing a dual-laser flow cytometer and data displayed as two-color dotplots. Each cytokine specific set of beads is assigned a uniquefluorescence intensity that is resolved on the FL-3 channel. Thepresence of each cytokine bound by the specific anti-IL4, -IL-6, -IL-10,-TNF-α, and -IFN-γ antibody coating the capture beads and detected byPE-conjugated anti-IL4, -IL-6, -IL-10, -TNF-α and -IFN-γ mAbs isindicated by the FL-2 signal intensity. Data are from one representativeexperiment out of five.

FIG. 2. CD160 mAb cross-linking triggers TNF-α, IFN-γ and IL-6 cytokineproduction by PB-NK cells. After cross-linking of CD160, CD16, NKG2D,and NK cell receptors by specific mAbs during 16 h incubation, samplesupernatants were analyzed by CBA for cytokine production, as describedin Materials and Methods. Cytokine concentrations in the samples werecalculated relative to the appropriate calibration curves with standarddilutions for each cytokine. Results are expressed as mean±SE of nineindependent experiments performed with different donors. *P≦0.05,**P≦0.03, ***P≦0.01, ****P≦0.003, *****P≦0.008 (Student T-test).

FIGS. 3A and 3B. Inhibition of CD160-mediated TNF-α, IFN-γ and IL-6cytokine production by the CD158b inhibitory receptor.

(FIG. 3A) Freshly purified PB-NK were immediately analyzed by flowcytometry for surface expression of CD160, CD56, CD3, CD16, CD158b, andNKG2D using PE-Cy5-conjugated BY55 anti-CD160 mAb and/or PE-conjugatedanti-CD56, -CD3, -CD16, -CD158b mAbs and/or anti-NKG2D mAb, followed byPE-conjugated F(ab′)₂ goat anti-mouse IgG1 Ab. Upper panel, singlestaining (dark profiles); open profiles are PE-Cy5-IgM or PE-IgG isotypecontrol staining. Lower panel, double staining: the percentage of cellspositive for both CD160 and another marker is indicated. Results arerepresentative of five different experiments.

(FIG. 3B) CD160, NKG2D, and CD158b NK cell receptors were cross-linkedalone or co-cross-linked on PB-NK cells with specific mAbs using theappropriate concentrations, as described in Materials and Methods.Following 16 h receptor activation, sample supernatants were analyzed byCBA, as described in Materials and Methods. Data are taken from onerepresentative experiment out of five performed with different donors.

FIGS. 4A, 4B, 4C, 4D. Effect of sHLA-G1 on VEGF or FGF2-induced ECproliferation, migration and capillary-like tube formation.

(FIG. 4A) Inhibition of VEGF-mediated HUVEC proliferation by sHLA-G1.Cells were seeded at low density in the presence of VEGF and incubatedwith varying concentrations of sHLA-G1 (sG1) or control sHLA-G1-β2mmonochain (sG1mono). After 7 days of culture, cells were trypsinized andcounted.

(FIG. 4B) Inhibition of VEGF-induced HUVEC migration by sG1 or sG1mono.Growth arrested HUVEC monolayers were scrapped and were either notstimulated (−) or stimulated with VEGF, in the absence (−) or in thepresence of sG1 or sG1mono. 18 h later cell monolayers were stained withMay-Grunwald Giernsa and the migration of cells was counted as indicatedin Mat. and Methods.

(FIGS. 4C and 4D) Inhibition of FGF-2-induced HUVEC in vitroangiogenesis by sHLA-G1. HUVEC were seeded on Matrigel diluted incollagen gel in the presence or absence of FGF-2 and/or sG1. 24 h later,photographs of each well was taken (FIG. 4C), and angiogenesis wasquantified as described in Materials and Methods (FIG. 4D).Photomicrographs of representative wells show the decreasedFGF-2-induced HUVEC tube formation after sHLA-G1 incubation, incomparison with FGF-2 alone or FGF-2 and control. The control forsHLA-G1 is culture supernatant from untransfected cells, passed throughimmunoaffinity column, eluted and pooled (10). Results in A, B and D aremeans±SD of triplicate wells and are representative of five independentexperiments.

FIGS. 5A, 5B. sHLA-G1 does not bind to VEGF receptors.

(FIG. 5A) HUVEC were incubated with ¹²⁵I-sHLA-G1 in the absence (−) orpresence of cold VEGF, FGF-2 or varying concentrations of sHLA-G1 (sG1).Unlabeled sHLA-G1 but not VEGF nor FGF-2 prevented ¹²⁵I-sHLA-G1 binding.

(FIG. 5B) HUVEC were incubated with iodinated VEGF in the presence ofcold sG1, FGF-2 or VEGF. Unlike cold VEGF, cold sG1 did not abrogateiodinated VEGF binding. Results are means±SD of triplicate wells and arerepresentative of 5 independent experiments.

FIGS. 6A, 6B, 6C. sHLA-G1 binds to the CD160 receptor expressed by EC.

(FIG. 6A) HUVEC were analyzed by flow cytometry after incubation withCD8, ILT2 or CL1-R2 (CD160) specific mAbs (open profiles) or Ig-isotypecontrol Ab (black profiles) followed by FITC-labeled conjugates, in thepresence or not of VEGF or sG1. Results are representative of sixindependent experiments.

(FIG. 6B) CD160 mRNA expression in NK92, HUVEC and PB-CD4+ lymphocyteswas measured by RT-PCR, using CD160 (top) or β-actin (bottom) primers.

(FIG. 6C) Predicted amino acid sequence alignment of CD160 expressed inNK92 (NK) and HUVEC.

(−) indicate identity.

FIGS. 7A, 7B. HLA-G tetramers bind to Jurkat-CD 160 and HUVEC.

(FIG. 7A, upper), By flow cytometry, mAb CL1-R2 stained Jurkat-CD160 butnot untransfected Jurkat (open profiles). Black profiles are Ig-isotypecontrol stainings.

(FIG. 7A, lower), HLA-G1 tetramer cross-linked with W6/32 mAb, followedby incubation with streptavidin-PE, binds to Jurkat-CD160 but not tountransfected Jurkat, whereas not-cross-linked HLA-G1 tetramer, followedby incubation with streptavidin-PE, binds to HUVEC (open profiles).Black profiles are control staining with streptavidin-PE.

(FIG. 7B) HUVEC were incubated or not with sHLA-G1 (100 ng/ml) at 4° C.After 2 h, cells were incubated with CL1-R2 mAb followed by PE-conjugateand analyzed by flow cytometry (open profiles). Black profile isIg-isotype control staining. Results are representative of 3 independentexperiments.

FIG. 8. mAb cross-linking of CD160 triggers inhibition of in vitroangiogenesis.

HUVEC were seeded on Matrigel diluted in collagen gel in the presence orabsence of FGF-2 and sHLA6G1 and/or mAb CD160 or Ig-isotype control. 24h later, photography of each well was taken and angiogenesis quantifiedas described in Materials and Methods. Results are mean+/6 SD oftriplicate wells and are representative of 5 independent experiments.

FIGS. 9A, 9B. Effect of hypoxia on EC CD160 expression.

HUVEC were incubated in normoxia or hypoxia (5% 02) conditions during 24h and analyzed for surface expression of CD160 using CL1-R2 mAb (FIG.9A), or VCMAM, using anti-CD106 mAb (FIG. 9B), followed by PE-labeledconjugate. Black profiles are Ig-isotype control stainings. Results arerepresentative of 3 independent experiments.

FIGS. 10A, 10B, 10C, 10D. CL1-R2 mAb immunohistochemistry on tumorsections, showing that CD160 is not expressed by tumor cells, but isexpressed at a high level by EC of lymphatic vessels at the periphery ofthe tumor and EC of microvessels inside the tumor.

(FIGS. 10A and 10B) CD160 staining of lymphatic microvessels at theperiphery of the tumor.

(FIGS. 10C and 10D) CD160 staining of microvessels inside the tumor.

FIG. 11. Induction of CD160 transcripts in CD4+ lymphocytes with IL-15.

FIG. 12 a, 12 b, 12 c, 12 d. sHLA-G1 inhibits VEGF- or FGF2-mediatedendothelial cell proliferation, migration and capillary-like tubeformation. (a) Proliferative response of HUVEC to VEGF. Effects ofrecombinant sHLA-G1 (sG1) or control sHLA-G1-□2m monochain (sG1mono).(b) Inhibition of VEGF-induced HUVEC migration by sHLA-G1. Growtharrested HUVEC monolayers were scraped and were either not stimulated(untreated) or stimulated with VEGF, in the absence (−) or in thepresence of sG1 or sG1mono. 16 h later cell monolayers were stained withMay-Grunwald Giemsa and the migration of cells was counted as indicatedin Methods. (c, d) sHLA-G1 inhibits FGF-2-induced angiogenesis. HUVECwere seeded on Matrigel in the presence or absence of FGF-2 and/or sG1.Photographs of each well were taken after 24 h. (c), and angiogenesiswas quantified as described in Materials and Methods (d). The control isculture supernatant from untransfected cells, passed throughimmunoaffinity column, eluted and pooled³⁴. ***P<0.001, ANOVA testResults in (a, b, and d) are means±SEM of triplicate wells and arerepresentative of five independent experiments.

FIG. 13 a, 13 b, 13 c, 13 d, 13 e. sHLA-G1 induces apoptosis ofendothelial cells. (a) Kinetics curve of apoptosis induction. SGHEC-7cells were incubated with conditioned media from PC3 cells transfectedwith sHLA-G1 (G1s) or empty vector (neo). Time lapse microscopy wascarried out to assess the appearance of apoptotic morphology. Mean±SEMof pooled data from seven experiments is shown. Although data wereobtained every 15 min, data points are only shown every 2 h for clarity.(b) Images of endothelial cells after treatment with sG1 or neoconditioned media (Supplementary FIG. 2 video clip online). (c) Kineticscurve of apoptosis induction by recombinant sHLA-G1 in the presence orabsence of the caspase inhibitor zVAD-fink assessed by time-lapsemicroscopy. Mean±SEM of pooled data from four experiments is shown. (d)The area under the curve was calculated from the kinetics curves shownin (c). **P<0.003 Mann Whitney U test. (e) Western blot analysis of p85cleaved PARP expression. SGHEC-7 cells were incubated in the absence(control) or presence of sHLA-G1.

FIG. 14 a, 14 b. sHLA-G1 does not interfere with VEGF receptors. (a)HUVEC were incubated with ¹²⁵I-sHLA-G1 in the absence (−) or presence ofcold VEGF, FGF-2 or varying concentrations of sHLA-G1 (sG1). UnlabeledsHLA-G1 but not VEGF nor FGF-2 prevented ¹²⁵I-sHLA-G1 binding. (b) HUVECwere incubated with ¹²⁵I-VEGF in the presence of cold sG1, FGF-2 orVEGF. Unlike cold VEGF, cold sG1 did not abrogate iodinated VEGFbinding. Results are means±SEM of triplicate wells and arerepresentative of three independent experiments.

FIG. 15 a, 15 b, 15 c. HUVEC express the CD160 receptor. (a) HUVEC andHMVEC were analyzed by flow cytometry after incubation with CD8, CD85d,CD85j or CL1-R2 (CD160) specific mAbs (open profiles) or Ig isotypecontrols (black profiles) followed by FITC-labeled conjugates. Resultsare representative of six independent experiments. (b) CD160 mRNAexpression by HUVEC. (c) Predicted amino acid sequence alignment ofCD160 expressed in HUVEC and NK92. (−) indicates identity.

FIG. 16 a, 16 b, 16 c, 16 d. Immunohistochemical staining of Lewis lungcarcinoma tumor sections with anti-CD160 mAb demonstrating CD160positive vessels in brown. Vessel network staining was localized at theperiphery of the tumor (a). Blood vessels in the periphery (b) and thecentre of the tumor (c,d) were also stained with CD160 mAb, whereastumor cells remained unstained. Magnification, ×400.

FIG. 17 a, 17 b, 17 c, 17 d. sHLA-G1 binds to the CD160 receptorexpressed by endothelial cells. (a, upper), anti-CD160 mAb stainsJurkat-CD160 but not untransfected Jurkat (black profiles, isotypecontrol). (a, lower), HLA-G1 tetramer binds to HUVEC and Jurkat-CD160control transfectant but not to untransfected Jurkat cells (blackprofiles, control staining with streptavidin-PE). (b) RecombinantsHLA-G1 blocks CD160 mAb binding to HUVEC (black profile, isotypecontrol). Results are representative of three independent experiments.(c) Soluble CL1-R2 anti-CD160 mAb triggers inhibition of in vitroangiogenesis. HUVEC were seeded on Matrigel in the presence or absenceof FGF-2 and sHLA-G1 and/or mAb CD160 (+++, 10 μg/ml; +, 1 μg/ml) orIgG1-isotype control (10 μg/ml). Photographs of each well were takenafter 24 h and angiogenesis quantified. Results are mean±SD oftriplicate wells and are representative of five independent experiments.***P<0.001, *P<0.005 by ANOVA test, compared to FGF-2-treated cells. (d)Soluble CL1-R2 anti-CD160 mAb induces endothelial apoptosis. SGHEC-7cells were incubated with CL1-R2 (+, 1 μg/ml, ++, 5 μg/ml, +++, 10μg/ml) or IgG1 isotype control (10 μg/ml) and time lapse microscopy wascarried out to assess the appearance of apoptotic morphology. Levels ofapoptosis after 50 h are shown with mean±SD of pooled data from 3experiments. *P<0.05, **P<0.01, ***P<0.001 by ANOVA test, compared tocontrol.

FIG. 18 a: Expression of CD160 on HUVEC and HMVEC as compared tonegative control cells (Smooth muscle cells and human fibroblast inprimary culture). Flow cytometry analysis using BY55 anti-CD160 mAb(IgM) as compared to IgM isotype control. Cells were incubated witheither of these antibodies, washed and incubated with an anti-IgM-FITCconjugate.

FIG. 18 b: CL-R2 mAb induces apoptosis of HUVEC but not of fibroblast(Assessment by annexin-V and PI double-staining flow cytometry)

FIG. 18 c: Same method as in FIG. 18 b. Mean of 2 different experiments(5 different wells for each experiment)

EXAMPLES Example 1 Engagement of CD160 by its HLA-C Physiological LigandTrimers a Unique Cytokine Profile Secretion in the Cytotoxic PeripheralBlood NK Cell Subset Materials and Methods

Cells. Effector cells were the human CD160⁺ NK92 line (ATCC NumberCRL-2407) cultured with IL-2 for several days, and fresh humanperipheral blood (PB)-NK cells derived from normal donors, purified byimmunomagnetic NK cell isolation kit (Miltenyi Biotec). PB-NK purity wasshown to be >90% CD3⁻ CD56⁺ by flow cytometry and >90% of purified PB-NKwere CD160⁺.

Two variants of human K562 erythroleukemia cells were used as targetcells: one variant (K562_(class I+)) expressed HLA-C when cultured withIFN-γ (publication under reference 14; ATCC CCL-243) whereas the other(K562 cultured with IFN-γ, ATCC CCL-243) did not K562-HLA-Cw5transfectants (K562-Cw5) were obtained by transfection of HLA-Cw5 cDNAin K562 MHC class I negative parental cells.

Antibodies and Flow Cytometry Analysis. mAbs Used Included:

-   -   CL1-R2 (anti-CD160 IgG1 ; hybridoma TM60 available from C.N.C.M.        Institut Pasteur 25, rue du Docteur Roux F-75724 PARIS CEDEX 15        FRANCE under C.N.C.M. deposit number=I-3204),    -   BY55 (anti-CD160 IgM; Beckman-Coulter),    -   W6/32 anti-HLA (IgG2a; ATCC Number HB-95), referred here as        anti-HLA-C,    -   PE-conjugated 3G8 anti-CD16 (IgG1; Beckman-Coulter),    -   GL183 anti-CD158b (IgG1; Beckman-Coulter),    -   anti-CD3 (UCHT1 from Beckman-Coulter)    -   anti-CD56 (Beckman-Coulter), and    -   anti-NKG2D clone 149810 (IgG1, R & D Systems).

For single staining flow cytometry analysis, cells were incubated withPE-Cy5-conjugated BY55 anti-CD160 mAb or with the other PE-conjugatedmAbs. For the NKG2D staining, cells were incubated with anti-NKG2D mAbfollowed by PE-5 conjugated F(ab′)₂ goat anti-mouse IgG1 Ab(Cliniscience). For double staining, cells were incubated withPE-Cy5-conjugated BY55, followed by PE-conjugated anti-CD56, -CD3,-CD16, -CD158b mAbs, or by anti-NKG2D mAb followed by PE-conjugatedF(ab′)₂ goat anti-mouse IgG1 Ab. PE-Cy5-IgM or PE-IgG (Beckman-Coulter)were used as isotype controls. Samples were analyzed on an EPICS XL4Cflow cytometer (Beckman-Coulter).

Receptor specific mAb-mediated cross-linking. Cross-linking of CD160,NKG2D, CD16, or CD158b receptors on PB-NK cells was performed in thefinal concentration of 1-10 μg/ml during 16 h incubation at 37° C. in 5%CO₂. IgG1 isotype control was also used at the same conditions. 100 U/mlIL-2 was added during the incubation time. Supernatants were collectedand stored at −80° C. until further analysis.

NK cells and CD160 ligand-expressing cells co-cultures. NK92 or PB-NKcells were incubated alone or co-incubated either with K562_(class I+),K562 or K562-Cw5 at a ratio of 10:1 during 4 h (NK92) or 16 h (PB-NK) at37° C. in the presence or not of blocking concentrations (25-50 μg/ml)of W6/32 or CL1-R2 mAbs or Ig-isotype controls. 100 U/ml IL-2 was addedduring the incubation times.

Intracellular TNF-α detection. NK92 cells treated as above were washed,fixed in 2% paraformaldehyde, permeabilized with 0.1% saponin for 10min, stained by PE-conjugated anti-TNF-αmAb or mouse IgG1-PE(Coulter-Immunotech) and analyzed by an EPICS XL4C flow cytometer(Coulter).

Cytokine measurement by Cytometric Bead Array. The Th1/Th2 CytometricBead Array (CBA) kit (BD Biosciences) was used for simultaneousmeasurement of IL-2, IL4, IL-6, IL-10, TNF-α and IFN-γ according to themanufacturer's instructions (Cook, E. B., J. L. Stahl, L. Lowe, R. Chen,E. Morgan, J. Wilson, R. Varro, A. Chan, F. M. Graziano, and N. P.Barney. 2001. Simultaneous measurement of six cytokines in a singlesample of human tears using microparticle-based flow cytometry:allergies vs. non-allergics. J. Immunol. Meth. 254:109-118.). Briefly,CBA uses a series of uniform-size beads with discrete fluorescenceintensity (FL3). Each series of beads is coated with a mAb against asingle cytokine (IL-2, IL-4, IL-6, IL-10, TNF-α or IFN-γ and the mixtureof beads detects six cytokines in one sample. A cytokine standardcontaining a mixture of predetermined amounts of all six cytokines wasused to prepare standard curves.10 μl aliquot of each capture beadspecific for IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ was mixed for eachassay tube to be analyzed. Then 50 μl of such mixed capture beads, 50 μlof human Th1/Th2-PE detection reagent and 50 μl of appropriate testsample (frozen supernatants from different treated PB-NK cells, thawedand centrifuged prior analysis) were added to each assay tube. Tubeswere incubated for 3 h at room temperature, washed and reconstituted in300 μl of wash buffer. Finally, IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γcytokine-bound cytometric beads were analyzed on a FACScalibur flowcytometer (Becton Dickinson) using CELLQuest (Becton Dickinson). Themean fluorescence was compared with standard curves and cytokineconcentrations (pg/ml) calculated by using the CBA software provided (BDBiosciences). IL-2 measurements were excluded from analysis because theculture medium in which NK cells were incubated during the differentassays always contained IL-2.

Statistics. Statistical analyses were performed using either thetwo-tailed Student-T test or Student paired-T test with p≦0.05 definedas significant.

Results and Discussion HLA-C Expressing K562 Target Cell Lines TriggerCytokine Production by PB-NK Cells

We investigated whether TNF-α production could be obtained in the NK92cell line which expresses high amount of CD160. Intracellular expressionof this cytokine was evaluated by flow cytometry in NK92 co-culturedwith HLA-C expressing K562 (K562_(class I+)) target cells. We found thatsuch co-culture stimulated TNF-α production, as compared with themoderate secretion of this cytokine by NK92 cultured alone (FIG. 1A).Absence of TNF-α production by K₅₆₂ _(class I+) alone indicated thatTNF-α release was produced solely by NK92. Furthermore, addition in theculture medium of mAb W6/32-mediated HLA-C-masking on target cellsresulted in a diminishment of TNF-α production by NK92 (FIG. 1A). Theseresults indicate that HLA-C was capable to trigger NK92 to secreteTNF-α.

Next, we evaluated whether cytotoxic PB-NK could also produce TNF-α uponspecific HLA-C-mediated triggering. PB-NK were co-cultured for 16 heither with K562_(class I+), K562-Cw5 transfectant, which both expressHLA-C molecules at their cell surface, or with K562 which is entirelyMHC class I negative (FIG. 1B). Using the CBA kit and flow cytometry,TNF-α and four other Th1/Th2 cytokines were measured in the cell-freesupernatant fluid (FIG. 1C for one representative experiment and Table Ifor 5 independent experiments).

TABLE I Table I IFN-γ, TNF-α and IL-6 production by PB-NK cellsco-cultured with HLA-C expressing K562 IFN- TNF- IL-4 IL- IL- Cells(pg/m (pg/m (pg/m (pg/m (pg/m N   185±  16± 0  16± 0 NK/K562_(Class-I+)29,441^(d)± 334^(b)± 0 620^(b)± 0 NK/K56   296±  19± 0  33± 0 NK/K562-20,089^(c)± 389^(b)± 0 689^(a)± 0 Purified PB-NK cells were culturedalone (NK) or co-cultured with K562_(Class-I+), K562, or K562-Cw5 cells.After 16 h, culture supernatants were collected and cytokineconcentrations measured by Cytometric Bead Array, as described inMaterials and Methods. Less than 10 pg/ml concentration was consideredas 0. Results are expressed as mean ± SE of five independent experimentsperformed with different donors which were selected according to theabsence of cytokine production when PB-NK were co-cultured with K562.^(a)P < 0.03, as compared to the NK group (Paired Student T-test). ^(b)P< 0.02 ^(c)P < 0.01 ^(d)P < 0.008

When PB-NK from different donors were co-cultured with K562_(class I+)or K562-Cw5, a large amount of IFN-γ, TNF-α and IL-6 was detected andneither IL-4 nor IL-10. By comparison, PB-NK co-cultured with class Inegative K562 produced very low amounts of IFN-γ and only marginalamounts of TNF-α and IL-6, not significantly different from thoseobserved when PB-NK cells were cultured alone (FIG. 1C and Table I). Nospontaneous cytokine release was ever produced when K562 orK562_(class I+) were cultured alone. However, we should mention that, insome donors, PB-NK did produce cytokines when co-cultured with K562.This suggested that MHC class I-independent activating receptors couldbe also involved. Altogether, these data indicate that HLA-Cphysiological ligand recognition by cytotoxic PB-NK cell subset couldtrigger specific cytokine secretion.

Specific Engagement of CD160 by Its Physiological Ligand HLA-C Resultsin IFN-γ, TNF-α and IL-6 Production by PB-NK

We investigated whether CD160 receptor triggered specific cytokinesecretion by PB-NK upon engagement with HLA-C. PB-NK were co-culturedwith K562_(class I+) in the presence of blocking concentrations of mAbsto either CD160 or HLA-C, or of Ig-isotype controls (Table II).

TABLE II Table II Anti-CD160 and -HLA-C blocking mAbs prevent productionof IFN-γ, TNF-α and IL-6 by PB-NK co-cultured with K562_(Class-I+) IFN-γTNF-α IL-4 IL-6 IL-10 Type (pg/m (pg/m (pg/m (pg/m (pg/m NK   186±  18±0  20± 0 NK/K562_(Class-I+) + 23,054± 200± 0 414± 0 IgG1NK/K562_(Class-I+) +  1,083^(a)±  58^(c)± 0 143^(b)± 0 anti-CD160 mAbNK/K562_(Class-I+) + 16,125± 215± 0 478± 0 IgG2a NK/K562_(Class-I+) + 1,149^(c)±  64^(c)± 0 370± 0 anti-HLA-C mAb Purified PB-NK cells werecultured alone (NK) or co-cultured with K562_(Class-I+) in the presenceof anti-CD160, or anti-HLA-C mAbs at blocking concentrations or Igisotype controls. After 16 h of incubation, culture supernatants werecollected and cytokine concentrations measured by Cytometric Bead Array,as described in Materials and Methods. Less than 10 pg/ml concentrationwas considered as 0. Results are expressed as mean ± SE of fourindependent experiments performed with different donors. ^(a)P < 0.04,as compared to the control NK/K562_(Class-I+) + IgG1 group (StudentT-test). ^(b)P < 0.03 ^(c)P < 0.01

Masking HLA-C ligand or CD160 receptor by their specific mAbssignificantly diminished the IFN-γ, TNF-α and IL-6 production. Theseresults show that this PB-NK cytokine production is mainly attributableto CD160-HLA-C interaction. However, for an unknown reason, the use ofW6/32 anti-HLA-C mAb did not significantly inhibit IL-6 secretion.

Antibody Cross-Linking of CD160 Expressed by Cytotoxic PB-NK Triggers aUnique Cytokine Production Profile Different from the One Obtained afterCD16 or NKG2D Engagement

We then compared the cytokines produced by CD160 triggering with theCD16 activating receptor whose expression is also restricted to thecytotoxic NK cell subset. The activating natural cytotoxic receptors(NCR) and 2B4/CD244 co-receptor were excluded from this comparison asthey are equally distributed on both cytotoxic and non-cytotoxic PB-NKlymphocytes (Ferlazzo, G., and C. Münz. 2004. NK cell compartments andtheir activation by dendritic cells. J. Immunol. 172:1333-1339.). NKG2Dactivating receptor triggering was used as negative control for itsinability to mediate cytokine production by itself in human NK cells(André, P., R. Castriconi, M. Espeli, N. Anfossi, T. Juarez, S. Hue, H.Conway, F. Romagne, A. Dondero, M. Nanni, S. Caillat-Zucman, D. H.Raulet, C. Bottino, E. Vivier, A. Moretta, and P. Paul. 2004.Comparative analysis of human NK cell activation induced by NKG2D andnatural cytotoxicity receptors. Eur. J. Immunol. 34:961-971.; Raulet, D.H. 2003. Roles of the NKG2D immunoreceptor and its ligands. Nat. Rev.Immunol. 3:781-790.). The results indicate that CD160-mAb cross-linkingleads PB-NK to produce the same pattern of cytokine release, namely highlevels of IFN-γ, and lower amounts of TNF-α and IL-6, but no IL-4 norIL-10 (FIG. 2), than the HLA-C physiological ligand triggering (Table1). The use of an isotype-matched control Ig did not lead to suchsecretion. Next, we analyzed the cytokine production after cross-linkingof CD16 receptor with the specific 3G8 mAb. This triggered both IFN-γand TNF-α production but no IL-6 (FIG. 2). Importantly, whereas theamount of TNF-α was comparable after CD160 or CD16 engagement, theproduction of IFN-γ mediated by CD16 cross-linking was ˜30 fold lessthan the secretion obtained after CD160 engagement. As expected, Abcross-linking of NKG2D did not trigger significant cytokine production.These data further demonstrate that Ab cross-linking of CD160 receptoron cytotoxic PB-NK cells results in a unique cytokine profile similar tothat observed after interaction with HLA-C physiological ligand. Itshould be of note that IL-6 production by a cytotoxic NK cell subset,upon triggering of activating receptors, has not been reported yet. IL-6is a multifunctional cytokine that acts in the immune system and arecent report has shown that some tumor-infiltrating lymphocytesproduced high concentrations of IL-6, blocking the anti-LAK activity oftumor cell TGF-β1 (Hsiao, Y. W., K. W. Liao, S. W. Hung, and R. M. Chu.2004. Tumor-Infiltrating Lymphocyte Secretion of IL-6 AntagonizesTumor-Derived TGF-beta1 and Restores the Lymphokine-Activated KillingActivity. J. Immunol. 172:1508-1514.).

Inhibition of CD160-Mediated NK Cell Cytokine Production by CD158bInhibitory Receptor

Activation of NK cells is dependent on activating receptors that arenormally functionally silenced by inhibitory receptors, including thekiller immunoglobulin-like receptors (KIRs) that recognize differentallelic groups of HLA-A, -B or -C molecules. We previously reported thatcytotoxic activity triggered upon CD160 engagement was inhibited by theco-engagement of CD158b inhibitory receptor (Le Bouteifler, P., A.Barakonyi, J. Giustiniani, F. Lenfant, A. Marie-Cardine, M.Aguerre-Girr, M. Rabot, I. Hilgert, F. Mami-Chouaib, J. Tabiasco, L.Boumsell, and A. Bensussan 2002. Engagement of CD160 receptor by HLA-Cis a triggering mechanism used by circulating natural killer (NK) cellsto mediate cytotoxicity. Proc. Natl. Acad. Sci. USA 99:16963-16968.). Wethus investigated whether inhibitory receptors also controlledCD160-mediated cytokine production We used PB-NK from donors who expressvariable percentages of cell population bearing CD158b inhibitoryreceptor. We analyzed cell surface expression of CD160, as well asCD158b, NKG2D and other NK cell markers by flow cytometry on freshlyisolated, purified PB-NK. FIG. 3A shows the results obtained with onerepresentative donor. A major subset of PB-NK expresses CD160, whereasall of them are CD56⁺, CD3⁻, and CD16⁺ (FIG. 3A, upper panel). Whereasthe whole PB-NK population is NKG2D⁺, only a subset expresses CD158binhibitory receptor. Double staining confirms that CD160⁺ PB-NK wereCD3⁻, and mostly CD56^(dim) and CD16⁺ (FIG. 3A, lower panel). Inaddition, we found that only subpopulations of CD160⁺ cells alsoexpressed CD158b or NKG2D (FIG. 3A, lower panel). As expected, we foundthat mAb-mediated cross-linking of CD160 and not of NKG2D receptor ledto the production of IFN-γ, TNF-α, and IL-6 and that, theco-cross-linking of both CD158b inhibitory receptor and CD160 reducedsignificantly the cytokine production (FIG. 3B). Such a reduction didnot occur when an isotype-matched control Ab substituted CD158b mAb.Similar results were obtained with five different PB-NK donors thatcontained variable percentages (˜8-30%) of CD158b⁺ NK subset among thepurified PB-NK cells. As only a sub-population of PB-NK did expressCD158b, it may explain why the down-modulation of cytokine secretion wasonly partial in our experiments. One can speculate that other KIRs,which interact with different HLA alleles, may also contribute to suchcontrol of CD160 inducing cytokine production and thus participate to NKcell tolerance in normal physiological situation. We also examinedwhether NKG2D co-engagement could synergize with CD160 to produce anaugmented stimulatory signal. We found that simultaneous cross-linkingof NKG2D, whose level is up-regulated following IL-2 activation, andCD160 activating receptors did not induce a cumulative positive signalcompared with stimulation through the CD160 receptor alone (FIG. 3B).This confirms previous results showing that human NKG2D triggering byspecific mAb cross-linking did not induce activation of cytokinesecretion (André, P., R. Castriconi, M. Espeli, N. Anfossi, T. Juarez,S. Hue, H. Conway, F. Romagne, A. Dondero, M. Nanni, S. Caillat-Zucman,D. H. Raulet, C. Bottino, E. Vivier, A. Moretta, and P. Paul. 2004.Comparative analysis of human NK cell activation induced by NKG2D andnatural cytotoxicity receptors. Eur. J. Immunol. 34:961-971.). However,stimulation of polyclonal activated NK cells with plastic-boundrecombinant MICA or ULBP physiological ligands could trigger GM-CSF andIFN-γ production (André, P., R. Castriconi, M. Espeli, N. Anfossi, T.Juarez, S. Hue, H. Conway, F. Romagne, A. Dondero, M. Nanni, S.Caillat-Zucman, D. H. Raulet, C. Bottino, E. Vivier, A. Moretta, and P.Paul. 2004. Comparative analysis of human NK cell activation induced byNKG2D and natural cytotoxicity receptors. Eur. J. Immunol. 34:961-971.).

CD160 receptor, whose expression is restricted to the effector cytotoxicCD56^(dim) CD₁₆ ^(bright) PB-NK cell subset, appears as a unique MHCclass I-dependent activating receptor capable to promote cytokinesecretion upon specific ligation. Firstly, HLA-C major ligand of CD160is constitutively expressed, which differs from the inducibleself-ligands or pathogens induced ligands of the other NK triggeringreceptors expressed on both cytotoxic and non-cytotoxic NK lymphocytesubsets. Human NKG2D ligands are the stress-induced MICA and MICBmolecules that are expressed predominantly by cells of epithelial originor pathogen encoded ULBP (Raulet, D. H. 2003. Roles of the NKG2Dimmunoreceptor and its ligands. Nat. Rev. Immunol. 3:781-790.). Inaddition, NKG2D is unable to trigger by itself IFN-γ production in human(Carayannopoulos, L., and W. Yokoyama 2004. Recognition of infectedcells by natural killer cells. Curr. Opin. Immunol. 16:26-33.). Therecently described Poliovirus receptor (CD155) and Nectin-2 (CD112)ligands of the DNAM-1 co-activating receptor are also mostly expressedin stressed tissues (Moretta, L., and A. Moretta 2004. Unravellingnatural killer cell function: triggering and inhibitory human NKreceptors. Embo J. 23:255-259.). NCR ligands are non-MHC molecules,including SV hemaglutinin-neuraminidase for NKp44 and NKp46(Carayannopoulos, L., and W. Yokoyama. 2004. Recognition of infectedcells by natural killer cells. Curr. Opin. Immunol. 16:26-33.). Incontrast to the above-mentioned receptors, CD16 is present only oneffector cytotoxic PB-NK lymphocyte subset and its ligand is the Fcportion of IgG. Secondly, stimulatory KIRs and CD94/NKG2C activatingreceptor that are only expressed by a subset of cytotoxic PB-NKlymphocytes, also interact with constitutive HLA class I molecules,including HLA-C for the former, have short cytoplasmic domains with noknown signaling motif (Cerwenka, A., and L. Lanier. 2001. Natural Killercells, viruses and cancer. Nat. Rev. Immunol. 1:41-49.). In addition,these activating receptors associate with adaptor molecules to initiatesignaling (Lanier, L. 2003. Natural killer cell receptor signaling.Curr. Opin. Immunol. 15:308-314.), which differs from CD160 GPI-anchoredcell surface molecule (Le Bouteiller, P., A. Barakonyi, J. Giustiniani,F. Lenfant, A. Marie-Cardine, M. Aguerre-Girr, M. Rabot, I. Hilgert, F.Mami-Chouaib, J. Tabiasco, L. Boumsell, and A. Bensussan. 2002.Engagement of CD160 receptor by HLA-C is a triggering mechanism used bycirculating natural killer (NK) cells to mediate cytotoxicity. Proc.Natl. Acad. Sci. USA 99:16963-16968.). 2B4/CD244 is an NK cell receptorthat provides a co-stimulatory signal to other activation receptorsincluding NCR or NKG2D (Moretta, L., M. Mingari, C. Bottino, D. Pende,R. Biassoni, and A. Moretta 2003. Cellular and molecular basis ofnatural killer and natural killer-like activity. Immunol. Letters88:89-93.).

Data from this study shows that stimulation of CD160 receptor on NKcells may lead to enhancement of both innate immunity (through specificcell killing) and adaptive immunity (through cytokine secretion).Strikingly, it has been shown that the HLA-C ligand of CD160 isprotected from degradation or endocytosis mediated by US2 or US11CMV-derived proteins (Tortorella, D., B. Gewurz, M. Furnan, D. Schust,and H. Ploegh. 2000. Viral subversion of the immune system. Ann. Rev.Immunol. 18:861-926.) or Nef HIV-1 proteins (Cohen, G., R. Gandhi, D.Davis, O. Mandelboim, B. Chen, J. Strominger, and D. Baltimore. 1999.The selective downregulation of class I Major Histocompatibility Complexproteins by HIV-1 protects HIV-infected cells from NK cells. Immunity10:661-671.), respectively. This suggests that CD160 may still befunctional soon after viral infection.

The signals that transform a circulating resting NK cell into anactivated cytokine-secreting cell in vivo are not fully understood. Thismainly depends on the outcome of signals derived from activating andinhibitory receptors upon engagement by their specific ligands. Knowingthat CD158a/CD158b inhibitory receptors engage HLA-C molecules on targetcells, we hypothesize that the level of expression of HLA-C may be a keyfactor to trigger either the KIR or CD160 receptors. When the level ofHLA-C is normal, KIR inhibitor receptor engagement would control CD160.In contrast, when the level of expression of HLA-C is down modulated,KIR receptors might no longer be efficiently engaged, allowing theactivating function of CD160 receptor to take place.

In conclusion, this study demonstrates that functional activation ofCD160 NK cell receptor by HLA-C physiological ligand initiates bothcytotoxicity and cytokine production after optimal receptor triggering.The present results strongly suggest that CD160 mediates the activatingeffector functions through a unique signaling pathway to limit viremiaand tumor burden or pathogen-infected cells.

Example 2 Cutting Edge: Soluble HLA-G1 Inhibits Angiogenesis Through theBinding to CD160 Receptor Expressed by Endothelial Cells

Material and Methods

Cells and Reagents

HUVEC and human microvascular endothelial cells (HMVEC) [HUVEC CC-2517,and neonatal HMVEC-C (CC-2505); Cambrex Bio Science, Walkersville, Md.,U.S.A; cf. http://www.cambrex.com/Content/bioscience/CatNav.oid.435]were maintained in EBM (BioWhittaker) supplemented with 5% FCS and 1ng/ml VEGF or FGF-2 (R & D systems, Minneapolis, Ill.) every other day.

Human Jurkat T Cells are Available from ATCC Number TIB152.

Jurkat cells transfected with CD160 (Jurkat-CD160) were produced bytransfection of CD160 in Jurkat cells as reported by Anumantha, A., A.Bensussan, L. Boumsell, A. Christ, R. Blumberg, S. Voss, A. Patel, M.Robertson, L. Nadler, and G. Freeman, 1998 (“Cloning of BY55, a novel Igsuperfamily member expressed on NK cells, CTL, and intestinalintraepithelial lymphocytes”, Journal of Immunology 161:2780.)

NK92 is a Human NK Cell Line Expressing CD160 (ATCC Number CRL-2407).

CD4⁺ T cells were purified from PBMC using the MACS separation system(Miltenyi Biotec, Auburn, Calif.). The sHLA-G1-β2m fusion monochain genewas engineered by connecting the last residue of the α3 domain of HLA-Gto the first codon of the human β2m sequence through a 15-residue spacer(Fournel, S., M. Aguerre-Girr, A. Campan, L. Salauze, A. Berrebi, Y.Lone, F. Lenfant, and P. Le Bouteiller. 1999. Soluble HLA-G:purification from eucaryotic transfected cells and detection by aspecific ELISA. American Journal of Reproductive Immunology 42:22.).sHLA-G1 and sHLA-G1mono were purified from eucaryotic cell culturesupernatants, using immunoaffinity columns, as previously described(Fournel, S., M. Aguerre-Girr, A. Campan, L. Salauze, A. Berrebi, Y.Lone, F. Lenfant, and P. Le Bouteiller. 1999. Soluble HLA-G:purification from eucaryotic transfected cells and detection by aspecific ELISA. American Journal of Reproductive Immunology 42:22.).VEGF 165 was expressed in a baculovirus system as described (Plouët J.,F. Moro, S. Bertagnolli, N. Coldeboeuf, H. Mazarguil, S. Clamens, and F.Bayard. 1997. Extracellular cleavage of the vascular endothelial growthfactor 189-amino acid form by urokinase is required for its mitogeniceffect. J Biol Chem 272:13390.).

mAbs Used Included:

-   -   CL1-R2 (anti-CD160 IgG1; hybridoma TM60 available from C.N.C.M.        Institut Pasteur 25, rue du Docteur Roux F-75724 PARIS CEDEX 15        FRANCE under C.N.C.M. deposit number=1-3204),    -   anti-CD8 (B9.11, Coulter Immunotech, Marseille, France),    -   anti-CD85j (BD Biosciences Pharmingen, San Diego, Calif., USA),    -   anti-CD106 (1G11 Coulter-Immunotech),    -   dialyzed mouse IgG1 or IgG2a isotype controls        (Coulter-Immunotech).

HLA-G tetramers were produced essentially as previously described(Allan, D. S., M. Colonna, L. L. Lanier, T. D. Churakova, J. S. Abrams,S. A. Ellis, A. J. McMichael, and V. M. Braud. 1999. Tetramericcomplexes of human histocompatibility leukocyte antigen (HLA)-G bind toperipheral blood myelomonocytic cells. J Exp Med 189:1149.), usingsynthetic self-peptide RIIPRHLQL (SEQ ID NO:7) and after addition ofstreptavidin-PE (Pharmingen) (Lee, N., A. R. Malacko, A. Ishitani, M. C.Chen, J. Bajorath, H. Marquardt, and D. E. Geraghty. 1995. Themembrane-bound and soluble forms of HLA-G bind identical sets ofendogenous peptides but differ with respect to TAP association. Immunity3:591). Labeling of HUVEC, Jurkat and Jurkat-CD160 by PE-conjugatedHLA-G tetramers was performed at 37° C. for 1 h. For Jurkat-CD160 andJurkat, tetramers were cross-linked with anti-class HLA class I W6/32mAb.

Lewis lung carcinoma cells are available from ECACC [European Collectionof Cell Cultures; Health Protection Agency, Porton Down; SP4 0JGSalisbury, Wiltshire UK] (human Caucasian lung carcinoma cell lineCOR-L23/R; deposit number ECACC 96042339).

Cell Proliferation and Migration Assays

For the proliferation analysis, HUVEC (8×10³) were seeded in 12-wellplates coated with 0.3% gelatin Cells were incubated with saline or VEGF(1 ng/ml) in the presence of absence of various concentrations ofsHLA-G1 or sHLA-G1mono. 7 days later, cells were trypsinized and countedin a Coulter counter ZM (Margency, France). Migration assays wereperformed on growth arrested confluent HUVEC or BAEC. Cell monolayerswere wounded with a rubber policeman. The dishes were washed withserum-free medium and each well was photographed at 100× magnification.Dishes were then incubated for 16 h in serum free medium containing ofsHLA-G1 or sHLA-G1mono (100 ng/ml) in the presence or not of VEGF (50ng/ml). A second photograph of each well was taken and the cells whichhad migrated were counted by superposing the two photographs.

Cell Binding of VEGF and sHLA-G1

VEGF and sHLA-G1 were iodinated with the iodogen procedure with aspecific activity of 240,000 and 110,000 cpm/ng, respectively (Plouët,J., F. Moro, S. Bertagnolli, N. Coldeboeuf, H. Mazarguil, S. Clamens,and F. Bayard. 1997. Extracellular cleavage of the vascular endothelialgrowth factor 189-amino acid form by urokinase is required for itsmitogenic effect J Biol Chem 272:13390.). Wells containing 2×10⁵serum-starved HUVEC were either pre-treated with 50 ng/ml VEGF orsHLA-G1 at 37° C. for various time intervals (0.1-24 h) or processedimmediately for binding assays. Briefly dishes were rinsed in cold DMEMsupplemented with 0.2% gelatin and 20 mM Hepes pH 7.3 and incubated at4° C. for 2 h with 2 ng/ml ¹²⁵I-VEGF or sHLA-G1 in the absence orpresence of unlabeled ligand. Cells were then rinsed in the same mediumand lysed in RIPA buffer and the radioactivity counted in a Packardgamma counter.

In Vitro Capillary Tube Formation

Growth factor reduced Matrigel (BD Biosciences) was diluted in collagen(1/6 v/v) and kept on ice. 160 μl of this solution was added to eachwell of 8-well culture slides precoated with type I rat tail collagenand left at 37° C. for 1 h. Following gel formation, a HUVEC suspension,mixed or not with control, FGF-2, sHLA-G1 or mAb CD160 was seeded onMatrigel/collagen gels for 24 h at 37° C. in a humidified 5% CO2incubator. Angiogenesis was quantified as previously described (RuggeriB, Singh J, Gingrich D, Angeles T, Albom M, Yang S, Chang H, Robinson C,Hunter K, Dobrzansld P, Jones-Bolin S, Pritchard S, Aimone L,Klein-Szanto A, Herbert J M, Bono F, Schaeffer P, Casellas P, Bourie B,Pili R, Isaacs J, Ator M, Hudkins R, Vaught J, Mallamo J, Dionne C.“CEP-7055: a novel, orally active pan inhibitor of vascular endothelialgrowth factor receptor tyrosine kinases with potent antiangiogenicactivity and antitumor efficacy in preclinical models”, Cancer Res. 2003Sep. 15;63(18):5978-91; Erratum in: Cancer Res. 2003 Nov. 1;63(21):7543.

Briefly, the culture medium was removed, cells rinsed twice with PBS andfixed for 30 min at room temperature in a 4% PFA solution. Then, thecells were washed twice with PBS and stained with Masson's Trichromstain. The extent of the microcapillary network was measured using anautomated computer-assisted image analysis system (Imagenia, Biocom, LesUlis, France), and the total length of the capillaries in each well wasdetermined. The mean microcapillary network length (μm) was calculatedfor each experimental condition. Experiments were performed intriplicate and repeated 3 times.

Flow Cytometry Analysis

Subconfluent HMVEC in normoxia or in hypoxia (24 hours incubated at 37°C. in a 5% O2 atmosphere) or HUVEC (Biowitthaker) were scrapped inPBS-BSA and incubated or not with 100 ng/ml of sHLA-G1 at 4° C. After 2h cells were incubated with either CD8, CD85d, CD85j (Plouët, J., F.Moro, S. Bertagnolli, N. Coldeboeuf, H. Mazarguil, S. Clamens, and F.Bayard. 1997. Extracellular cleavage of the vascular endothelial growthfactor 189-amino acid form by urokinase is required for its mitogeniceffect. J Biol Chem 272:13390.1), CD106 (BD), CL1-R2 BY55 (Fournel, S.,M. Aguerre-Girr, A. Campan, L. Salauze, A. Berrebi, Y. Lone, F. Lenfant,and P. Le Bouteiller. 1999. Soluble HLA-G: purification from eucaryotictransfected cells and detection by a specific ELISA. American Journal ofReproductive Immunology 42:22.) specific mAbs or isotypic control Abs 20μg/ml followed by F(ab′)2-FITC conjugated goat anti-mouse IgG. Nonviable cells were excluded by the use of propidium iodide. Cells wereanalyzed by a Coulter-Epics ELITE flow cytometer.

RT-PCR and cDNA Sequencing

CD160 transcripts were detected by RT-PCR using the following primers:5′-3′ (sense) TGCAGGATGCTGTTGGAACCC (SEQ ID NO:1) and 3′-5′ (reverse)TCAGCCTGAACTGAGAGTGCCTTC (SEQ ID NO:2; cDNA quality was confirmed byamplification of β actin using the following primers: 5′-3′GCGGGAAATCGTGCGTGCGTGACA (SEQ ID NO:3) and 3′-5′GATGGAGTTGAAGGTAGTTTCGTG (SEQ ID NO:4). Amplification conditions forCD160 and β-actin were 95° C. for 45 s, 60° C. 30 s, and 72° C. for 1min, for 35 cycles. For CD160 sequencing, a Taq High Fidelity was used(Invitrogen). PCR product was purified (qiaex II, Qiagen) and analyzedwith the following primers:

BY01 (5′-3′ sense) (TGCAGGATGCTGTTGGAACCC; SEQ ID NO: 1), BY03(3′-5′ reverse) (TCAGCCTGAACTGAGAGTGCCTTC; SEQ ID NO: 2; BY02(5′-3′ sense) CAGCTGAGACTTAAAAGGGATC; SEQ ID NO: 5) and BY04(3′-5′ reverse) (CACCAACACCATCTATCCCAG; SEQ ID NO: 6).

Syngenic Tumor for Histological Studies

Sub-confluent Lewis lung carcinoma cells were trypsinized, washed twiceand resuspended in PBS. 2.10⁵ cells were inoculated subcutaneously intothe right posterior lateral flank of anaesthetised (pentobarbital, IP)female C57B16 mice (IFFA CREDO, France). Mice were killed 21 days aftercell injection with an overdose of pentobarbital; tumors were removedand fixed in 10% neutral buffered formalin (Sigma) overnight at 4° C.,paraffin embedded (Embedder Leica) and then sectioned (5 μm) with amicrotome (Leica). After rehydration (toluene/ethanol/PBS), slides wereheated for 20 min in a citrate buffer solution at pH 6.1. Sections wereplaced in a DAKO Autostainer and incubated with TNB Blocking buffer (TSAKit, NEN), peroxidase-blocking reagent (Dako) and Mouse on Mouseimmunoglobulin blocking reagent (Vector Laboratories). Tumours vesselswere stained with the monoclonal antibody CL1-R2 at a finalconcentration of 10 μg/ml (Dilution 1/500 de la solution) during 30 minat room temperature. Sections were then incubated with biotin-labelledgoat anti-rabbit IgG for 10 min followed by incubation withAvidin-Biotin Complex (Vector Laboratories) for 30 min. Sections werethen stained with DAB (Vector Laboratories) and counterstained withhematoxylin. Immunostained tissues were viewed on a Nikon microscope(E-800) and digitised using a DMX 1200 camera (Nikon) with 40×objective.

Results

sHLA-G1 Inhibits VEGF- or FGF2-Induced Endothelial Cell Proliferation,Migration and Capillary-Like Tube Formation

VEGF is the more potent mitogenic and motogenic factor for vascular EC.Therefore we investigated whether sHLA-G1 could interfere with VEGFfunctions on EC in vitro. We found that sHLA-G1 inhibited VEGF-inducedproliferation of HUVEC (FIG. 4A) whereas it did not affect basalproliferation of these cells. In contrast, when sHLA-G1 was fused toβ2m, the single chain protein did not affect the proliferation of ECinduced by VEGF, therefore suggesting that folding of the molecule wascritical for its biological activity. Moreover, sHLA-G1 inhibitedVEGF-induced proliferation of bovine EC derived from aorta or adrenalgland microvessels, suggesting a mechanism conserved among species andorgan of origin of the EC.

In a migration assay using HUVEC as endothelial cell model no migrationoccurred whether or not sHLA-G1 or sHLA-G1mono were added in the absenceof VEGF (FIG. 4B). In contrast, after addition of VEGF, a significantincrease in the number of migrated cells was detected. In theseconditions, addition of sHLA-G1 inhibited migration, whereas sHLA-G1mono had no significant effect (FIG. 4B). To evaluate whether sHLA-G1was able to block tube formation after stimulation by pro angiogenicfactors, HUVEC were subjected to FGF-2 in the Matrigel model. For thispurpose, the Matrigel was diluted with collagen to limit spontaneousangiogenesis which normally occurs after 3 days in culture. Morphologyof the cells in Matrigel is shown in FIGS. 4C and the quantification ofthe total tubules length is shown in FIG. 4D. The results indicate thatFGF-2 induced a potent angiogenic response and that addition of sHLA-G1to FGF-2 significantly inhibited tube formation.

Altogether, these results demonstrate that sHLA-G1 is able to inhibitpro-angiogenic factor-induced EC proliferation, migration and in vitrovessel formation.

sHLA-G1 Did Not Interfere with VEGF Receptors

In this study, both ¹²⁵I-VEGF and ¹²⁵I-sHLA-G1 were used as ligands.Total binding of radiolabelled ligands to HUVECs cells at 4° C. was timedependent and reached equilibrium 45 min after the beginning of theexperiment After 60 min, incubation with unlabelled ligand almosttotally dissociated ¹²⁵I-VEGF or ¹²⁵I-sHLA-G1 binding from theendothelial cells. Thus, equilibrium binding experiments were performedby setting the incubation time at 60 min. Whatever the radio-ligandused, total binding was dose-dependent and non-specific binding,measured in the presence of a high concentration of unlabelled ligands,was linear with the concentration of radioligand. The non-specificbinding did not exceed 20% of the total binding. In competitionexperiments using ¹²⁵I-sHLA-G1 as ligand, while sHLA-G1 rapidlydisplaced the binding to HUVEC with IC50 values in a nanomolar range,VEGF preincubated or not with the cells did not affect this binding(FIG. 2A). In competition experiments using now ¹²⁵I-VEGF as ligand,while VEGF rapidly displaced the binding of ¹²⁵I-VEGF to HUVEC with IC50values in a nanomolar range, sHLA-G1 pre-incubated or not with the cellsdid not affect this binding (FIG. 2B). These results demonstrate thatsHLA-G1 was able to bind specifically to endothelial cells and that thisbinding was not modulate by VEGF. Moreover we clearly demonstrates thatsHLA-G1 did not interfere with the VEGF receptors on endothelial cells.

CD160 Receptor is Expressed By Endothelial Cells

Using specific mAbs and flow cytometry analysis, we found that HUVEC didnot express CD8, nor CD85j. In contrast, these cells were stronglystained by an anti-CD160 mAb (FIG. 6A) like HMVEC. To provide additionalevidence that CD160 was expressed by HUVEC, we performed RT-PCR analysison these cells by comparison to CD160+(NK92) and CD160⁻ (CD4+ T) controlcells, using CD160 specific primers. Similarly to NK92, CD160 mRNA wasdetected in HUVEC, whereas CD4+ T cells were negative (FIG. 6B). ThenHUVEC and NK92 cDNAs were isolated and sequenced. Predicted amino acidsequence alignment of HUVEC and NK92 CD160 proteins showed that theywere both similar to the CD160 sequence already described (Anumantha,A., A. Bensussan, L. Boumsell, A. Christ, R. Blumberg, S. Voss, A.Patel, M. Robertson, L. Nadler, and G. Freeman. 1998. Cloning of BY55, anovel Ig superfamily member expressed on NK cells, CTL, and intestinalintraepithelial lymphocytes. Journal of Immunology 161:2780.) (FIG. 6C).Altogether these data demonstrate that CD160 was expressed by EC.

sHLA-G1 Interacts With CD160 Expressed at the Cell Surface of HUVEC

Having shown that CD160 was present on EC, we investigated whethersHLA-G1 could be a potential ligand. The direct interaction of CD160with sHLA-G1 on HUVEC was demonstrated by using HLA-G1 tetramers. Wefirst showed that these tetramers specifically bound to Jurkat-CD160,but not to untransfected Jurkat (FIG. 7A), demonstrating the specificityof CD160 for sHLA-G1 ligand. When HUVEC were incubated with HLA-G1tetramers, a clear staining was detected, suggesting that sHLA-G1 boundto CD160 expressed by this cell (FIG. 7A). CD160-sHLA-G1 interaction wasfurther evaluated by flow cytometry on HUVEC which were pre-incubated ornot with sHLA-G1. We found that pre-incubation of HUVEC with sHLA-G1down-modulated CD160 cell surface expression (FIG. 7B). Thisdemonstrates that sHLA-G1 directly interacts with CD160 at the cellsurface of HUVEC.

mAb Cross-Linking of CD160 Expressed By Endothelial Cells TriggersInhibition of Capillary-Like Tube Formation

Next, we investigated whether CD160-mAb cross-linking could mimic thesHLA-G1 anti-angiogenic activity. Using the in vitro Matrigelassay, wefound that CD160-mAb cross-linking leads to the inhibition ofFGF2-mediated tubule vessel growth (FIG. 8). These data furtherdemonstrated that CD160, expressed by EC was a functional receptor ableto trigger an anti-angiogenic cell response.

Hypoxia Induced an Increase in the Expression of CD160 on EndothelialCells

While many of the individual phenotypic process in angiogenesis such ascell migration or endothelial tube formation can be induced by hypoxicculture conditions, we determined the expression of CD160 in HMVECcultured under hypoxic conditions (Luttun, A., M. Autiero, M. Tjwa, andP. Carmeliet. 2004. Genetic dissection of tumor angiogenesis: are PIGFand VEGFR-1 novel anti-cancer targets? Biochim Biophys Acta 1654:79.).Using specific CD160 mAb and flow cytometry analysis, we found thathypoxia strongly increased CD160 expression on endothelial cells (FIG.9).

Immunohistochemical Staining of LLC Tumors Demonstrate That Only ECExpressed CD160 in the Tumor

Finally, we observed a strong staining for CD160 on EC in LLC tumors(FIGS. 10A, 10B, 10C, 10D) whereas no staining was observed withnon-specific IgG. Tumors cells did not express CD160 but EC of lymphaticvessels at the periphery of the tumor or microvessels inside the tumorsexpress a high level of CD160.

Discussion

In this study, we identified a new receptor, CD160, able to trigger ananti-angiogenic response in endothelial cells. We demonstrated, for thefirst time, that this MHC class I-dependent receptor is expressed by EC.

CD160 triggers inhibition of VEGF- or FGF-2-induced in vitroangiogenesis upon engagement with its physiological ligand, sHLA-G1, orfollowing specific mAb cross-linking (CL1-R2).

CD160 differs from the previously described CD36 receptor, atransmembrane glycoprotein bound by thrombospondin 1 (TSP-1), a potentinhibitor of angiogenesis (Dawson, D. W., S. F. Pearce, R. Zhong, R. L.Silverstein, W. A. Frazier, and N. P. Bouck. 1997. CD36 mediates the Invitro inhibitory effects of thrombospondin-1 on endothelial cells. JCell Biol 138:707.). In contrast to CD36, CD160 is a GPI-anchoredmolecule having no transmembrane domain, nor cytoplasmic tail(Anumantha, A., A. Bensussan, L. Boumsell, A. Christ, R. Blumberg, S.Voss, A. Patel, M. Robertson, L. Nadler, and G. Freeman. 1998. Cloningof BY55, a novel Ig superfamily member expressed on NK cells, CTL, andintestinal intraepithelial lymphocytes. Journal of Immunology161:2780.).

We further found that sHLA-G1 was an EC CD160 ligand. Knowing thatvarious HLA class I molecules may bind to CD160 (Le Bouteiller, P., A.Barakonyi, J. Giustiniani, F. Lenfant, A. Marie-Cardine, M.Aguerre-Girr, M. Rabot, I. Hilgert, F. Mami-Chouaib, J. Tabiasco, L.Boumsell, and A. Bensussan. 2002. Engagement of CD160 receptor by HLA-Cis a triggering mechanism used by circulating natural killer (NK) cellsto mediate cytotoxicity. Proc Natl Acad Sci U S A 99:16963.; Agrawal,S., J. Marquet, G. J. Freeman, A. Tawab, P. Le Bouteiller, P. Roth, W.Bolton, G. Ogg, L. Boumsell, and A. Bensussan. 1999. Cutting edge: MHCclass I triggering by a novel cell surface ligand costimulatesproliferation of activated human T cells. J Immunol 162:1223.), othersoluble MHC class I molecules may also trigger this receptor to exertanti-angiogenic functions. We indeed observed that a recombinant solubleHLA-B7 could also inhibit HUVEC proliferation. These observationssuggest that the anti-angiogenic function of sHLA-G1 and sHLA-B7 couldbe generalized to all soluble HLA.

The anti-angiogenic activity of sHLA-G1 reported here is the first nonimmune function described to date. Spatial and temporal regulation ofangiogenesis at the materno-fetal interface plays an important role inensuring adequate blood supply to nourish the developing embryo,suggesting that there are local acting factors that regulate vasculargrowth (Ong, S., G. Lash, and P. N. Baker. 2000. Angiogenesis andplacental growth in normal and compromised pregnancies. Baillieres BestPract Res Clin Obstet Gynaecol 14:969.). sHLA-G1 is secreted byextravillous trophoblast, including endovascular trophoblast (Morales,P. J., J. L. Pace, J. S. Platt, T. A. Phillips, K. Morgan, A. T.Fazleabas, and J. S. Hunt. 2003. Placental cell expression of HLA-G2isoforms is limited to the invasive trophoblast phenotype. J Immunol171:6215.) that replaces EC of the maternal spiral arteries, therebyincreasing by several fold the diameter of these vessels (Loke, Y., andA. King. 2000. Immunology of implantation. Baillire's ClinicalObstetrics Gynaecology 14:827.). We hypothesize that sHLA-G1anti-angiogenic effect might contribute to such replacement Lack ofHLA-G expression in preeclamptic placentas, characterized by a shallowcytotrophoblast invasion and a reduced flow of maternal blood to thefeto-placental unit (Lim, K. H., Y. Zhou, M. Janatpour, M. McMaster, K.Bass, S. H. Chun, and S. J. Fisher. 1997. Human cytotrophoblastdifferentiation/invasion is abnormal in pre-eclampsia Am J Pathol151:1809.), favors such hypothesis.

Hypoxia has been shown to regulate the expression of multiple angiogenicendothelial markers as CD54, CD105 or tie-2 receptor. Over-expression oftie-2 suggests that it is involved in a positive angiogenic response tohypoxia. Up-regulation of CD160 by hypoxia could be generate a negativeregulation of angiogenesis and could prevent neovessels formation.Moreover, immunohistochemical on mouse tumor with CD160 antibody showsthat this receptor is not expressed by tumor cells themselves but isexpressed by EC of the tumoral vasculature. All these resultsdemonstrate that CD160, up-regulated by hypoxia, is an inhibitorysignaling receptor for angiogenesis and that its activation may beuseful for experimental anti-angiogenic therapy to prevent tumoral cellgrowth.

Example 3 CD160 is Not Restricted to the Cytotoxic T and NK Subset Butis Also Expressed By CD4+ T Cells

Freshly isolated peripheral blood (PB)-CD4+ cells were obtained fromlymphocytes of normal individual using the immunomagnetic CD4 cellisolation kit (Miltenyi Biotec). PB-CD4+ purity was shown to be >98%CD3+CD4+CD8− by flow cytometry. PB-CD4+ were further cultured forseveral days (between 3 to 6 days) in a standard culture mediumcontaining 10% of heat inactivated human AB serum supplemented with ahigh concentration of IL-15. The CD160 transcripts were detected byRT-PCR using the following primers:

(SEQ ID NO: 8) 5′-3′ (sense): TGCAGGATGCTGTTGGAACCC; (SEQ ID NO: 9)3′-5′ (reverse): TCAGCCTGAACTGAGAGTGCCTTC.

illustrative results are shown on FIG. 11.

Example 4 Assessment of Apoptosis By Annexin-V and PI Double-StainingFlow Cytometry

Material and Methods

0.2×10⁶ cells were seeded into a 6 wells/plate, serum-starved for 24 hand then treated with sHLA-G1 (1 μg/ml), CL1-R2 mAb CD160 (10 μg/ml) orcontrol Ig-G1 (10 μg/ml) for 50 h in the presence of VEGF (50 ng/ml). Atthe end of the treatment, the floating cells were collected bycentrifugation, whereas adherent cells were harvested by trypsin-EDTAsolution to produce a single cell suspension. The cells were thenpelleted by centrifugation and washed twice with PBS. Apoptotic celldeath was identified by double staining with recombinant FITC(fluorescein isothiocyanate)-conjugated Annexin-V and PI (propidiumiodide), using the Annexin V-FITC Apoptosis Detection kit (DAKO)according to manufacturer's instructions. Cells were analyzed by flowcytometry on a FACScan (Becton Dickinson) using the fluorescence 1 (FL1)signal detector for FITC conjugates and the FL3 signal detector for PI.Teen thousand events were recorded for each sample. The data wereanalyzed using CellQuest software.

Results

FIG. 18 a: demonstrates that CD160 receptor is expressed on the cellsurface of HUVEC and HMVEC but not of human fibroblast in primaryculture nor smooth muscle cells.

FIG. 18 b: demonstrates that CL1-R2 triggers specific apoptosis of HUVECand not fibroblasts.

FIG. 18 c: indicate that the CL1-R2 anti-CD160 monoclonal antibodymimics the anti-angiogenic effect of the soluble HLA-G1 natural ligandof CD160. Both soluble HLA-G1 as well as the anti-CD60 monoclonalantibody mediate endothelial cell (HUVEC) specific apoptosis, the IgG1isotype control being inefficient Accordingly, Annexin V bindingexperiments establish the specificity of this effect: the CL1-R2monoclonal antibody induces apoptosis of CD160 expressing HUVEC but notof CD160 negative primary fibroblast.

Example 5 Soluble HLA-G1 Inhibits Angiogenesis Through Apoptotic Pathwayand By Direct Binding to CD160 Receptor Expressed By Endothelial Cells

Abstract

HLA-G is a Major Histocompatibility Complex class Ib molecule whoseconstitutive tissue distribution is mainly restricted to trophoblastcells at the maternal-fetal interface during pregnancy. In this study wedemonstrate the ability of soluble HLA-G1 (sHLA-G1) isoform to inhibitvascular endothelial growth factor-induced endothelial cellproliferation and migration, and to decrease fibroblast growthfactor-2-induced capillary-like tube formation. We identify potentialmechanisms by which this occurs: sHLA-G1 induces apoptosis throughbinding to CD160, a glycosylphosphatidylinositol-anchored receptorexpressed by endothelial cells. Furthermore, we show that the specificCL1-R2 anti-CD160 monoclonal antibody mimics sHLA-G1-mediated inhibitionof endothelial cell tube formation and induction of apoptosis. Thus,engagement of CD160 in endothelial cells may be essential for theinhibition of angiogenesis. sHLA-G1/CD160-mediated anti-angiogenicproperty may participate in the vascular remodeling of maternal spiralarteries during pregnancy, and offers an attractive therapeutic targetto prevent pathologic neovascularization as we found that CD160 isstrongly expressed in the vasculature of a murine tumor.

Introduction

HLA-G is a human major histocompatibility class Ib gene characterized bya unique promoter region, limited polymorphism, restricted constitutivetissue distribution and the occurrence of several spliced transcriptsencoding either membrane-bound or soluble proteins¹. The activelysecreted soluble HLA-G1 (sHLA-G1) isoform derives from mRNA retainingintron 4², which contains a stop codon that precludes translation of thetransmembrane domain. This 37 kDa intron-retaining sHLA-G1 isoformassociates with β2-microglobulin (β2m)². The predominant expression ofsHLA-G1 in the placenta at a time when polymorphic HLA-A and HLA-B classIa molecules are repressed is consistent with important immunologicalfunctions during pregnancy³. sHLA-G1 induces apoptosis of activated CD8+T and NK cells^(4,5) and down-regulates CD4+ T cell allo-proliferationresponse⁶. The observation that some anti-HLA-G monoclonal antibodies(mAb) bound to placental endothelial cells7,8 led to our hypothesis thatHLA-G might also be involved in the modulation of placental angiogenesisand uterine vessel remodeling⁷. Several further observations are in linewith such a novel function of HLA-G: first, a defect of HLA-G expressionin extravillous cytotrophoblast is associated with preeclampsia^(9,10),a common complication of pregnancy in which HLA-G⁺ endovasculartrophoblast invasion of maternal spiral arteries is abrogated,compromising blood flow to the maternal interface⁹; second, it has beenshown that HLA-G inhibited the transendothelial migration of NK cells¹¹and the rolling adhesion of activated NK cells on endothelial cells¹².To date, the potential role of sHLA-G1 in the modulation of angiogenesishas not been addressed.

Angiogenesis, the formation of new capillaries from preexisting bloodvessels, is a crucial component of embryonic vascular development anddifferentiation, wound healing, and organ regeneration, but it alsocontributes to the progression of pathologies that depends onneovascularization, including tumor growth, diabetes, ischemic oculardisease, and rheumatoid arthritis¹³⁻¹⁵. While the most importantmediators of angiogenesis, the endothelial growth factor (VEGF) and thefibroblast growth factor (FGF) families are well defined¹⁶, angiogenesisexists as a complex process involving multiple gene products expressedby different cell types, all contributing to an integrated sequence ofevents¹⁷.

To test our hypothesis that sHLA-G1 is a regulator of endothelial cellactivity, we investigated its in vitro effect. This study demonstratesthat sHLA-G1 inhibits VEGF- and FGF-induced angiogenesis and inducesapoptosis of endothelial cells by interaction with theglycosylphosphatidylinositol (GPI)-anchored CD160 receptor18,19expressed on endothelial cells. Interestingly, we show byimmunohistochemistry performed ex vivo that CD160 is expressed at thevascular level in a mouse tumor model.

Results

sHLA-G1 Inhibits VEGF- or FGF2-Mediated Endothelial Cell Proliferation,Migration and Capillary Tubule Formation

VEGF is the most potent mitogenic and motogenic factor for endothelialcells¹⁶. Therefore, we investigated whether sHLA-G1 could interfere withVEGF functions in vitro. Purified recombinant sHLA-G1, when addedexogenously to HUVEC, inhibited the proliferative response to VEGF in adose-dependent manner (FIG. 12 a). In contrast, the single chain proteinsHLA-G1 fused to β2m (sHLA-G1mono) had no effect, indicating thatfolding of sHLA-G1 was critical for its biological activity. Moreover,sHLA-G1 also inhibited VEGF-mediated proliferation of bovine endothelialcells derived from aorta or adrenal gland microvessels (data not shown),suggesting a mechanism conserved among species and endothelial cellsoriginating from different tissues.

We then examined the effect of sHLA-G1 on endothelial cell migration,using a migration assay. When HUVEC were incubated in the absence ofVEGF, marginal, spontaneous migration occurred, whether or not sHLA-G1was added (FIG. 12 b, untreated). In contrast, after addition of VEGF, asignificant increase in the number of migrated cells was detected. Underthese conditions, addition of sHLA-G1 inhibited their migration (FIG. 12b, VEGF-treated). The inhibition of migration was not observed withsHLA-G1 mono.

We next evaluated the capacity of sHLA-G1 to inhibit capillary tubuleformation by endothelial cells cultured on Matrigel. sHLA-G1significantly inhibited FGF-2-induced tube-like formation (FIG. 12 c,morphology and FIG. 12 d, quantification). Collectively, these findingsindicate that sHLA-G1 inhibits in vitro pro-angiogenic factor-mediatedendothelial cell proliferation, migration, and capillary tube formation.

sHLA-G1 Induces Apoptosis of Endothelial Cells

In order to determine the mechanisms involved in these sHLA-G1-inducedinhibitory effects, we examined, using time lapse microscopy, whetherapoptotic morphological changes occurred in endothelial cells aftersHLA-G1 treatment. Using HUVEC-derived endothelial cells, we found thatincubation of these cells with conditioned medium containing sHLA-G1clearly induced apoptosis, as determined by time-lapse digital imagemicroscopy (FIG. 13 a, b). Images from the end of the experiment (FIG.13 b) and video data show that sHLA-G1-treated cell morphology ischaracterized by cytoplasmic and nuclear shrinkage and a change to aphase bright appearance, as well as the formation of membraneblebs/blisters. By comparison, incubation of these cells in controlconditioned medium had no effect. Experiments in which recombinantsHLA-G1 (100 ng/ml) was used showed similar apoptotic effects (FIG. 13c,d). Use of the broad-spectrum caspase inhibitor zVAD-fmk preventedrecombinant sHLA-G1-mediated apoptosis (FIG. 13 c,d). Induction ofapoptosis by sHLA-G1 was further demonstrated by the detection ofcleaved poly (ADP-ribose) polymerase (PARP) by Western blot analysis(FIG. 13 e).

sHLA-G1 Binds Directly to the CD160 Receptor

Next, it was important to identify the receptor involved in thesHLA-G1-mediated inhibitory effects on endothelial cells. We firsttested whether sHLA-G1 interfered with VEGF receptors, by performingradioreceptor-assay binding experiments at 4° C. on HUVEC incubated for2 hours (equilibrium time) with ¹²⁵I-VEGF or ¹²⁵I-sHLA-G1 in thepresence of various concentrations of cold competitors. We firstanalyzed the binding of ¹²⁵I-sHLA-G1 to HUVEC and found that cold VEGFor FGF-2 competitors had no effect, whereas unlabeled sHLA-G1 inhibitedthis binding as a function of the concentration with IC50 values in thenanomolar range (FIG. 14 a). In competition experiments using ¹²⁵I-VEGFas ligand, we found that cold VEGF rapidly displaced its binding toHUVEC with IC50 values in a nanomolar range, whereas sHLA-G1 had noeffect (FIG. 14 b). Furthermore, we found that cold sHLA-G1 competitordid not inhibit the binding of ¹²⁵I-VEGF to PAEC-VEGF-R2 or PAEC-NPL1transfectants (data not shown). However other inhibitors ofVEGF-dependent proliferation and migration, such as dopamine²⁰, may actthrough internalization of VEGF receptors without competing with VEGFcell binding. Pre-incubation of HUVEC with VEGF at 37° C. almost totallyabolished the binding of ¹²⁵I-VEGF within 1 h, whereas that of sHLA-G1up to 24 h had no effect on its binding (data not shown), thusdemonstrating that it did not modulate the internalization or theexpression of VEGF receptors. These results demonstrate that sHLA-G1bound specifically to endothelial cells without interfering with VEGFreceptors.

Then we investigated whether HUVEC expressed some of the HLA-G receptorsdescribed to date, including CD8⁴, CD85d²¹, CD85j²¹, and CD160²². Flowcytometry analysis revealed that HUVEC were stained by anti-CD160 mAb,although not with constant levels, but not by anti-CD8, -CD85d nor-CD85j mAbs (FIG. 15 a). Similarly, HMVEC also bound anti-CD160 mAb(FIG. 15 a), as did bovine endothelial cells (data not shown),suggesting that the CD160 epitope recognized by the mAb was conservedamong species. To further demonstrate that CD160 is expressed by HUVEC,we performed RT-PCR analysis on these cells by comparison to CD160⁺ (NKcell line NK92) and CD160⁻ (CD4⁺ T) control cells, using CD160 specificprimers. Similarly to NK92, CD160 mRNA was detected in HUVEC, whereasCD4⁺ T cells were negative (FIG. 15 b). Then HUVEC and NK92 cDNAs wereisolated and sequenced. Predicted amino acid sequence alignment of HUVECand NK92 CD160 proteins showed that they were both similar to the CD160sequence already described¹⁹, with the exception of two substitutedresidues indicating a possible allelic form (FIG. 15 c).

To demonstrate that CD160 is also expressed on endothelial cells in vivoand that its expression could not result from culture conditions, weperformed a specific immunohistochemical staining of a grafted Lewislung carcinoma mouse tumor. We found that the anti-CD160 mAb stronglystained endothelial cells of micro vessels at the periphery of (FIG. 16a,b) and inside the tumor (FIG. 16 c,d), whereas no staining wasdetected with IgG isotypic control (data not shown). In contrast, tumorcells remained unstained. Such reactivity of the CL1-R2 anti-CD160 mAbis not surprising as the previous identification and sequencing of bothhuman and mouse CD 160 encoding cDNA revealed a strong homology betweenthe two species¹⁹.

We then demonstrated that sHLA-G1 did effectively bind to the CD160receptor expressed by endothelial cells. We first found that a HLA-G1tetramer specifically bound to HUVEC like it did on CD160-transfectedJurkat (FIG. 17 a). sHLA-G1-CD160 direct interaction on HUVEC wasfurther demonstrated by showing that pre-incubation of HUVEC withrecombinant sHLA-G1 specifically blocks the binding of anti-CD160 mAb(FIG. 17 b), whereas a pre-incubation with VEGF did not (data notshown).

To further demonstrate that sHLA-G1 function is mediated throughinteraction with CD160, we tested whether soluble anti-CD160 mAb couldmimic the sHLA-G1 anti-angiogenic activity in the in vitro Matrigelassay and the pro-apoptotic effect. The results clearly showed thataddition of 1 to 10 μg/ml of purified CL1-R2 mAb led to the inhibitionof FGF-2-mediated tubule vessel growth (FIG. 17 c) and induction ofendothelial apoptosis (FIG. 17 d). These data further demonstrate thatCD160 expressed by endothelial cells is a functional receptor able totrigger an anti-angiogenic cell response.

Discussion

In this study, we demonstrated that the sHLA-G1 molecule could exert anon immune function, namely angiogenesis inhibition. Spatial andtemporal regulation of the vasculature at the maternal-fetal interfaceplays an important role in ensuring adequate blood supply to nourish thedeveloping embryo, suggesting that there are locally acting factors thatregulate vascular cells²³. sHLA-G1 is secreted by extravilloustrophoblast, including endovascular trophoblast³ that replacesendothelial cells and remodels the maternal spiral arteries, therebyincreasing the diameter of these vessels several fold²⁴. We hypothesizethat sHLA-G1 effects on endothelial cells might contribute to suchreplacement. Defects of HLA-G expression, including diminishment ofsoluble HLA-G in preeclamptic placentas, characterized by a shallowcytotrophoblast invasion and a reduced flow of maternal blood to thefeto-placental unit^(9,10), favors such hypothesis.

Different mechanisms have been reported to explain the activity ofangiogenesis inhibitors, including induction of endothelial cellapoptosis²⁵, inhibition of matrix metalloproteinase activity²⁶, orchemorepulsion of endothelial cells²⁷. In this report we demonstratenovel inhibitory actions of sHLA-G1, including significant blockade ofendothelial cell migration, proliferation and vessel formation. Inaddition, we suggest that these effects may involve induction ofendothelial cell apoptosis since sHLA-G1-treated endothelial cellsprogressively showed apoptotic morphology. Whether this apoptosis ismediated by endothelial FasL expression, like in activated CD8⁺ Tcells⁴, remains to be demonstrated. It is interesting that a role forapoptosis and Fas/FasL interactions in the remodeling of uterinearteries during pregnancy has recently been demonstrated²⁸.

The direct inhibitory effect of sHLA-G1 on vessel formation is mostlikely mediated through the functional CD160 receptor, as the CL1-R2anti-CD160 mAb mimics the inhibition of FGF-2-induced capillary tubuleformation by endothelial cells cultured in Matrigel and the induction ofendothelial apoptosis. In contrast to other angiogenesis inhibitors likesemaphorin 3F which is a competitor of VEGF binding to neuropilinreceptor²⁷, sHLA-G1 acts directly on CD160 receptor. Knowing thatvarious HLA class I molecules may bind to CD160²⁹, it cannot be excludedthat other soluble MHC class I molecules could also trigger thisreceptor to exert anti-angiogenic functions. Collectively these findingsprovide important mechanistic insights into anti-angiogenic action ofsHLA-G1. Further investigation is needed to determine the signalingpathways used by endothelial cells and NK cells following CD160engagement and leading to apoptosis for the former and cytokineproduction³⁰ and cytotoxicity²⁹ for the latter.

In addition to the clear importance in the placental/uterineenvironment, the identification of CD160 as an inhibitory signalingreceptor for angiogenesis could be useful for experimentalanti-angiogenic therapy to prevent tumor cell growth. Ourimmunohistochemical analysis of a mouse graft tumor showed that CD160,encoded by a gene conserved in this species³¹, was present inendothelial cells of the tumoral vasculature but was not expressed bytumor cells. Future goals are therefore to examine the potentialCD160/sHLA-G1 mediated anti-angiogenic effect in different tumors andexplore the possible therapeutic use of CD160 in the regulation ofpathological neovascularization.

Methods

Cells and reagents. Human umbilical vein endothelial cells (HUVEC) andhuman microvascular endothelial cells (HMVEC) (BioWhittaker, San Diego,Calif.) were maintained in EBM (BioWhittaker) supplemented with 5% FCSand 1 ng/ml VEGF or FGF-2 (R & D systems, Minneapolis, Ill.) every otherday. SGHEC-7 cells are a HUVEC-derived cell line, cultured as previouslydescribed³². Porcine aortic endothelial cell (PAEC)-VEGF-R2 (KDR),PAEC-NPL1 transfectants, human Jurkat T cells and Jurkat transfectedwith CD160 (Jurkat-CD160)²⁹ were produced locally. NK92 is a human NKcell line expressing CD160²⁹. CD4⁺ T cells were purified from PBMC usingthe MACS separation system (Miltenyi Biotec, Auburn, Calif.). Prostateadenocarcinoma PC3 cells transfected with PCDNA vector containing intron4-retaining sHLA-G1 cDNA and PC3 cells transfected with empty vector(PC3-neo)³³ were grown to confluence for 4 days and conditioned mediacollected. Media was removed, centrifuged to remove cell debris andstored at −20° C. The sHLA-G1-β2m fusion monochain gene was engineeredby connecting the last residue of the α3 domain of HLA-G to the firstcodon of the human β2m sequence through a 15-residue space³⁴. sHLA-G1and sHLA-G1mono were purified from eukaryotic cell culture supernatants,using immunoaffinity columns, as previously described³⁴. VEGF 165 wasexpressed in a baculovirus system as described³⁵. mAbs used includedCL1-R2 (IgG1) anti-BY55/CD160²⁹, produced in one of our laboratories,anti-CD8 (OKT8, Coulter Immunotech), anti-ILT4/CD85d (gift of M.Colonna), anti-ILT2/CD85j, anti-CD106 (Beckton Dickinson), and dialyzedmouse IgG1 or IgG2a isotype controls (DAKO or Sigma). HLA-G1 tetramerswere produced essentially as previously described³⁶, using syntheticself-peptide RIIPRHLQL³⁷ and after addition of streptavidin-PE(Pharmingen). Labeling of HUVEC, Jurkat and Jurkat-CD160 byPE-conjugated HLA-G tetramers was performed at 37° C. for 1 h. ForJurkat-CD160 and Jurkat, tetramers were cross-linked with anti-HLA classI W6/32 mAb, as previously described²².

Endothelial cell proliferation and migration assays. For theproliferation analysis, HUVEC were seeded into 12-well plates (8,000cells/well) coated with 0.3% gelatin in PBS. Cells were incubated withsaline or VEGF (1 ng/ml) in the presence or absence of variousconcentrations of sHLA-G1 or sHLA-G1mono. Seven days later, cells weretrypsinized and counted using a Coulter counter ZM. Migration assayswere performed on growth arrested confluent HUVEC or BAEC. Cellmonolayers were wounded with a rubber policeman, washed with serum-freemedium and each well was photographed at 100× magnification. Dishes werethen incubated for 16 h in serum free medium containing of sHLA-G1 orsHLA-G1mono (100 ng/ml) in the presence or not of VEGF (50 ng/ml). Asecond photograph of each well was taken and the cells which hadmigrated were counted by superposing the two photographs.

VEGF and sHLA-G1 cell binding. Purified recombinant VEGF and sHLA-G1were radiolabelled with Na¹²⁵I to a specific activity of 2,4×10⁴ and1,1×10⁵ cpm/ng, respectively³⁵. Wells containing 2×10⁵ serum-starvedHUVEC were either pre-treated with 50 ng/ml of VEGF or sHLA-G1 at 37° C.for various time intervals (0.1-24 h) or processed immediately forbinding assays. Briefly, dishes were rinsed in cold DMEM supplementedwith 0.2% gelatin and 20 mM Hepes (pH 7.3) and incubated at 4° C. for 2h with 2 ng/ml ¹²⁵I-VEGF or ¹²⁵I-sHLA-G1 in the absence or presence ofindicated concentrations of cold competitors. Cells were then rinsed inthe same medium, lysed in RIPA buffer and radioactivity counted in a γcounter.

In vitro capillary tube formation. Growth factor reduced Matrigel (BDBiosciences) was diluted in collagen (1/6 v/v) and kept on ice. 160 μlof this solution was added to each well of 8-well culture slidesprecoated with type I rat tail collagen and left at 37° C. for 1 h. AHUVEC suspension, mixed or not with control, FGF-2, sHLA-G1 or mAb CD160was seeded into Matrigel/collagen gels for 24 h at 37° C. Microtubuleswere quantified by microscopy as previously described³⁸. Briefly, theculture medium was removed, cells rinsed twice with PBS and fixed for 30min at room temperature in a 4% PFA solution. Then, the cells werewashed twice with PBS and stained with Masson's Trichrome. The extent ofthe microcapillary network was measured using an automatedcomputer-assisted image analysis system (Imagenia, Biocom), and thetotal length of the capillaries in each well was determined. The meanmicrocapillary network length (μm) was calculated for each experimentalcondition. Experiments were performed in triplicate and repeated threetimes.

Flow cytometry analysis. Sub confluent HUVEC or HVMEC were scraped inPBS-EDTA and incubated in the presence or absence of 100 ng/ml ofsHLA-G1 at 4° C. After 2 h, cells were incubated with anti-CD8, -CD85d,-CD85j, -CD106 or CL1-R2 anti-CD160 specific mAbs or Ig-isotype control(20 μg(ml) followed by F(ab′)₂-FITC- or PE-conjugated anti-mouse IgG.Non-viable cells were excluded by the use of propidium iodide. Sampleswere analyzed on a Coulter-Epics ELITE flow cytometer.

RT-PCR and cDNA sequencing. CD160 transcripts were detected by RT-PCRusing the following primers: 5′-TGCAGGATGCTGTTGGAACCC-3′ (SEQ ID NO: 8)and 3′-TCAGCCTGAACTGAGAGTGCCTTC-5′ (SEQ ID NO: 9). cDNA quality wasconfirmed by amplification of β-actin using the appropriate primers.Amplification conditions for CD160 and β-actin were 95° C. for 45 s, 60°C. 30 s, and 72° C. for 1 min. For CD160 sequencing, a Taq High Fidelitywas used (Invitrogen). PCR product was purified (qiaex II, Qiagen) andanalyzed with the following primers:

BY01 (5′-TGCAGGATGCTGTTGGAACCC-3′ (SEQ ID NO: 8)), BY03(3′-TCAGCCTGAACTGAGAGTGCCTTC-5′ (SEQ ID NO: 9)), BY02(5′-CAGCTGAGACTTAAAAGGGATC-3′ (SEQ ID NO: 5)) and BY04(3′-CACCAACACCATCTATCCCAG-5′ (SEQ ID NO: 6)).

Immunohistochemistry. Sub-confluent Lewis lung carcinoma cells weretrypsinized, washed twice and suspended in PBS. 2×10⁵ cells wereinjected subcutaneously into the dorsal midback region of C57BL/6 femalemice (IFFA Credo, France). Tumors were taken on day 21, fixed with 10%formalin (Sigma) overnight at 4° C., and embedded in paraffin (EmbederLeica). 5 μm sections were placed in a Dako Autostainer and incubatedwith TNB blocking buffer (TSA kit, NEN), peroxidase-blocking reagent(DAKO) and mouse immunoglobulin blocking reagent (Vector Laboratories).Sections were incubated with CL1-R2 anti-CD160 mAb (10 μg/ml), followedby biotin-labeled goat anti-mouse IgG and avidin-biotin complex (VectorLaboratories). They were stained with DAB (Vector Laboratories),counterstained with hematoxylin, viewed on a Nikon microscope (E-800)and digitized using a DMX 1200 camera (Nikon) with 40× objective.

Time-lapse microscopy. SGHEC-7 cells were seeded into 6-well plates(2.5×10⁵ cells/well in normal culture medium. After 15 h, conditionedmedia from PC3-sG1 or PC3-neo cells, recombinant sHLA-G1 (100 ng/ml),CL1-R2 anti-CD160 mAb (1-10 μg/ml), IgG1 isotype control (10 μg/ml) orzVAD-fink (50 μmol/l, Calbiochem) were added to the wells. The plate wastransferred to an Olympus IX70 inverted fluorescence microscope withmotorized stage and cooled CCD camera and enclosed in a heated,humidified chamber at 37° C. with 5% CO₂ in air. Images were taken every15 min for 36-50 h and time-lapse sequences were analyzed using ImageProPlus (Media Cybernetics). In each field of view 40 cells were randomlychosen. The experiments were repeated at least four times. Apoptoticcells were scored according to the time at which clear apoptoticmorphology was first observed³⁹.

Western blot analysis of cleaved PARP expression. SGHEC-7 endothelialcells were seeded in culture plates. After 16 h the cells werestimulated with recombinant sHLA-G1 (100 ng/ml) for 60 h. Cells werelysed in RIPA buffer with 0.1 mg/ml PMSF, 30 μl/ml aprotinin, and 1mmol/l sodium orthovanadate at 4° C. for 30 min. The samples wereseparated by SDS-PAGE and transferred to a nitrocellulose membrane.Following incubation in blocking buffer for 1 h at room temperature, themembrane was incubated with rabbit polyclonal anti-human cleaved PARP(Promega) for 1 h. Anti-rabbit IgG peroxidase (A6154, Sigma) was addedfor 1 h. Detection of membrane bound antibodies was carried out bychemiluminescence (ECLPlus, Amersham).

Statistical analysis. Results are expressed as mean±SEM or SD of “n”independent experiments and assessed using the Mann Whitney U test orANOVA test as appropriate and Everstat or GraphPadPrism software withP<0.05 considered statistically significant.

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1-53. (canceled)
 54. Drug comprising Anti-CD160 compound selected fromthe group consisting of anti-CD160 mAb CL1-R2 obtainable from thehybridoma deposited as CNCM I-3204, a conservative fragment of said mAbCL1-R2 and a conservative derivative of said mAb CL1-R2 comprising atleast one CL1-R2 fragment, wherein said anti-CD160 compound is capableof competing with the anti-CD160 mAb CL1-R2 for binding to CD160, and issufficiently CD160-specific for binding to CD160 without binding to atleast CD8αβ.
 55. Drug according to claim 54, wherein said anti-CD160compound does further not bind to CD85j.
 56. Drug according to claim 54,wherein said anti-CD160 compound is a conservative fragment of said mAbCL1-R2 selected from the group consisting of a Fab, a Fab′, a F(ab)₂, aF(ab′)₂ and a Fv fragment of said mAb CL1-R2.
 57. Drug according toclaim 54, wherein said anti-CD160 compound is a conservative derivativeof said mAb CL1-R2, which comprises at least one scFv compoundcomprising at least one CL1-R2 VH region of CL1-R2 linked to at leastone CL1-R2 VL region of CL1-R2 via a peptide linker (L).
 58. Drugaccording to claim 57, wherein said conservative derivative is amonovalent scFv.
 59. Drug according to claim 57, wherein saidconservative derivative is a multivalent scFv.
 60. Drug according toclaim 54, wherein said anti-CD160 compound is a conservative derivativeof said mAb CL1-R2 which comprises a scFv multimer derived from saidCL1-R2 mAb, joined to a Fc fragment.
 61. Drug according according toclaim 54, wherein said anti-CD160 compound is a conservative derivativeof said mAb CL1-R2 which comprises at least one Fv fragment of CL1-R2linked to a human Fc.
 62. Drug according to claim 54, wherein saidanti-CD160 compound is a conservative derivative, of said mAb CL1-R2obtainable by adding one or more Fab derived from said CL1-R2 mAb at theC-terminus of each H chain of the full length CL1-R2 mAb.
 63. Drugaccording to claim 54, wherein said anti-CD160 compound is aconservative derivative of said mAb CL1-R2 obtainable by covalentlylinking full-length CL1-R2 mAbs together to form an aggregated Ab form.64. Drug according to claim 54, wherein said anti-CD160 compound is aconservative derivative of said mAb CL1-R2 obtainable by linking two ormore Fabs head-to-tail.
 65. Drug according to claim 54, wherein saidanti-CD160 compound further comprises an immunotoxin and/or aradioelement.
 66. Drug according to claim 54, wherein said anti-CD160compound is a soluble compound.
 67. Drug according to claim 54, whereinsaid anti-CD160 compound is a compound comprising at least one CD160binding site and at least one CD158b binding site.
 68. Drug according toclaim 54, wherein said anti-CD160 compound is an aggregated compound.69. Drug according to claim 68, wherein said aggregated compoundcomprises a least three CD160 binding sites and no CD158b binding site.70. A method of treating angiogenesis in an individual comprisingadministering to said animal an anti-CD160 drug composition according toclaim
 54. 71. The method of claim 70, wherein said anti-angiogenic drugis administered for the prevention or treatment of a tumor.
 72. Themethod of claim 70, wherein said anti-angiogenic drug is administeredfor the prevention or treatment of pre-eclampsia or eclampsia.
 73. Themethod of claim 70, wherein said anti-angiogenic drug is administeredfor the prevention or treatment diabetes, and/or an ischemic oculardisease, and/or rheumatoid arthritis.
 74. A method of inducing orup-regulating the adaptive immunity potential of an individualcomprising administering to said individual an anti-CD160 composition asdefined in claim
 68. 75. The method of claim 74, wherein said drug isadministered for the treatment or prevention of an infection.
 76. Themethod of claim 74, wherein said drug is part of a vaccine composition,or is provided as a vaccine adjuvant.
 77. A method of inducing orup-regulating hematopoiesis in an individual comprising administering tosaid individual an anti-CD160 composition of claim
 68. 78. A method ofinducing or up regulating an inflammatory reaction in an individualcomprising administering to said individual an effective amount of ananti-CD160 composition of claim
 68. 79. A method of inhibiting ordown-regulating the adaptive immunity potential of an individualcomprising administering to said individual an anti-CD160 composition ofclaim
 66. 80. A method of inhibiting or down-regulating hematopoiesis inan individual comprising administering an anti-CD 60 composition ofclaim
 66. 81. A method of inhibiting or down-regulating an inflammatoryreaction in an individual comprising administering to said individual ofan anti-CD160 composition of claim
 66. 82. A method of treating B-cellchronic lymphocytic leukaemia in an individual comprising administeringto said subject an anti-CD160 composition of claim 54.